Site-specific incorporation of amino acids into molecules

[0001] This invention was made with federal government support under the grant number GM62523, awarded by NIH. The United States Government has specific rights to this invention.

BACKGROUND OF THE INVENTION [0002] Protein engineering is a powerful tool for modifying the structural catalytic properties and binding of natural proteins, and for de novo design of artificial proteins. Protein engineering relies on an effective recognition mechanism for the introduction of mutated amino acids in desired protein sequences. Although this process is very useful in the design of new macromolecules with precise control of composition and structure, the main limitation is that mutagenesis is limited to 20 naturally occurring amino acids. However, it is becoming increasingly clear that by introducing unnatural amino acids, the scope and impact of protein engineering methods can be expanded.

[0003] Unnatural amino acids having a variety of new functional groups have been globally exchanged for residue-specific replacement or introduction into recombinant proteins. Biosynthetic assimilation of non-canonical amino acids to proteins was achieved largely by using the ability of the wild-type synthesis apparatus to use analogs of naturally occurring amino acids (Budisa 1995, Eur. J. Biochem 230: 788-796; Deming 1997, J. Macromol. Sci. Pure Appl. Chem A34; 21432150; Duewel 1997, Biochemistry 36: 3404-3416; van Hest and Tirrell 1998, FEBS Lett 428 (12): 68-70; Sharma et al., 2000, FEBS Lett 467 (1): 37-40). However, there are situations when substitution or introduction of a single site by unnatural amino acids is needed. Such a method would allow the protein to be adjusted (size, acidity, nucleophilicity, hydrogen bonds or hydrophobic properties, etc. of the amino acids) to meet the specific structural and functional properties of interest. The possibility of locally introducing such amino acid analogues into proteins would significantly expand our ability to rationally and systematically manipulate the structure of proteins, both to study protein function and to create proteins with new properties. For example, the ability to synthesize large amounts of proteins containing heavy atoms will facilitate the determination of the protein structure, and the ability to site-specific replacement of fluorophores or photo-cleavable proteins in living cells would be powerful tools for studying the function of proteins in vivo.

[0004] In recent years, several laboratories have continued to increase the number of genetically encoded amino acids, both with the help of a nonsense suppressor

-2tRNA or suppressor tRNA shifted reading frame to incorporate non-canonical amino acids into proteins in response to amber or tetrabasic codons, respectively (Bain et al., J. Am. Chem. Soc. 111: 8013, 1989; Noren et al., Science 244 : 182, 1989; Furter, Protein Sci. 7: 419, 1998; Wang et al., Proc. Natl. Acad. Sci. USA, 100: 56, 2003; Hohsaka et al., FEBS Lett. 344: 171: 1994 ; Kowal and Oliver, Nucleic Acids Res. 25: 4685, 1997). Such methods insert non-canonical amino acids at codon positions that normally inhibit wild-type peptide synthesis (e.g., a stop codon or reading frame shift mutation). These methods proved effective for single-site insertion of new amino acids. However, their utility for substitution or introduction specific to many positions (compared to residue specific) is limited by moderate (20-60%) suppression efficiencies (Anderson et al., J. Am. Chem. Soc. 124: 9674, 2002; Bain et al., Nature 356: 537, 1992; Hohsaka et al., Nucleic Acids Res. 29: 3646, 2001). This is partly because too high stop codon suppression efficiency will interfere with the normal translation termination of some undirected proteins in the body. On the other hand, low suppression efficiency is likely to be insufficient to suppress more than one nonsense site or mutation shifting the reading frame in the target protein, so that the synthesis of a full length target protein containing more and more non-canonical amino acids becomes increasingly difficult or impractical.

[0005] Effective multi-site insertion has been achieved by replacing natural amino acids in Escherichia coli auxotrophic strains, for example, using aminoacyl-tRNA synthetases with free substrate specificity or altered editing activity (Wilson and Hatfield, Biochim. Biophys. Acta 781: 205, 1984; Kast and Hennecke, J. Mol. Biol. 222: 99, 1991; Ibba et al., Biochemistry 33: 7107, 1994; Sharma et al., FEBS Lett. 467: 37, 2000; Tang and Tirrell, Biochemistry 41: 10635, 2002; Datta et al., J. Am. Chem. Soc. 124: 5652, 2002; Doring et al., Science 292: 501, 2001). Although this method ensures the efficient introduction of analogues in many places, it suffers from such a limitation that the new amino acids must "share" codons together with one of the natural amino acids. So for each codon position in which both natural and new amino acids can be inserted, apart from the probability of insertion, there is relatively little control over which ultimately the amino acid will insert into. This can be a disadvantage, because for a modified enzyme or protein, the introduction of a non-canonical amino acid at a random location may unexpectedly compromise the function of the protein, while the lack of introduction of a non-canonical amino acid at the designed site will result in the project's goal not being achieved.

[0006] Generally, multi-site substitution methods are relatively simple to perform, but all sites corresponding to a given natural amino acid in the entire protein are replaced. The degree of introduction of natural and non-naturally occurring amino acids may also be variable. Furthermore, multi-site introduction of analogs often causes toxicity when cells are used, making it difficult to study the mutated protein in living cells. The present invention

-3 overcomes these obstacles by allowing a site-specific mutation of amino acids in proteins.

[0007] Some embodiments disclosed herein provide a new technique for introducing replacement amino acids, including naturally occurring amino acids or atypical or non-canonical amino acids into proteins, which is based on a break in the degeneracy of the genetic code. In particular, certain embodiments of the present invention allow site-specific substitution or introduction of unnatural amino acids into proteins with high fidelity.

[0008] US2004 / 0053390 (Datta et al.) Discloses methods, reagents and calculation tools for the design of unnatural enzyme substrate analogues, especially for the design of unnatural amino acid analogs for aminoacyl-tRNA synthetases such as Phe-tRNA synthetase.

BRIEF SUMMARY OF THE INVENTION [0009] Some embodiments disclosed herein provide component compositions used in protein biosynthesis machines that include external mutated aminoacyl-tRNA synthetase (AARS) molecules optionally paired with external mutated aminoacyl-tRNA molecules.

[0010] Also provided are methods for creating and selecting external mutant tRNAs, external mutant aminoacyl-tRNA synthetases, and pairs thereof that are capable of introducing amino acids, including unnatural amino acids into peptides and proteins. Certain compositions of specific embodiments include new mutant external tRNA or external mutated aminoacyl tRNA synthetase pairs. New external mutant tRNA molecules, AARS molecules or AARS-tRNA pairs can be used to introduce an unnatural amino acid into a polypeptide in vitro and in vivo. Other embodiments of the invention include selecting external mutant pairs.

[0011] The compositions of the present invention comprise an outer mutated aminoacyl-tRNA synthetase, wherein the outer mutant tRNA synthetase preferably aminoacylates the mutated outer tRNA with an unnatural amino acid, optionally, in vivo. In one embodiment, a nucleic acid or polynucleotide encoding a mutated external synthetase, or a complementary nucleic acid sequence thereof, is provided.

[0012] Thus, some embodiments include a composition comprising a first vector comprising a polynucleotide encoding a modified aminoacyl yeast phenylalanyl-tRNA synthetase (PheRS), said polynucleotide being mutated at the codons encoding the amino acid at sequence codons selected from the group consisting of amino acid sequence number numbers (i) 412 and 415; (ii) 415 and 418; (iii) 415 and 437; (iv) 412, 415 and 437; (v) 415, 418 and 437; (vi) 412, 415 and 418; and (vii) 412, 415, 418 and 437 of the polypeptide encoded by SEQ ID NO: 3 and wherein said modified synthetase is capable of attaching an unnatural amino acid to a tRNA molecule.

[0013] At least one embodiment further comprises a second vector comprising a polynucleotide encoding a tRNA molecule. In at least one embodiment, said first and second vectors are the same vector. In other embodiments, said first and second vectors are different vectors.

[0014] In at least one embodiment, the tRNA is endogenous, and in at least one embodiment, the tRNA is modified. In at least one embodiment, the tRNA is modified such that it contains a mutated anti-codon that undergoes base pairing with an appropriate degenerate wobble codon with an affinity greater than that of natural tRNA. In some embodiments, the AARS and tRNA are from the same or different organisms. In at least one embodiment, the unnatural amino acid is selected from the group consisting of: azidororleucine, 3- (1-naphthyl) alanine, 3- (2-naphthyl) alanine, p-ethynylphenylalanine, p-propargyl-oxy-phenylalanine, m-ethynyl-phenylalanine, 6-ethynyltryptophan, 5-ethynyl tryptophan, acid ( R) -2-Amino-3- (4-ethynyl-1H-pyrrol-3-yl) propane, pbromophenylalanine, 77-idiophenylalanine, p-azidophenylalanine, 3- (6-chloroindolyl) alanine, 3- (6-bromoindolyl) alanine, 3- (5-bromoindolyl) alanine, azidohomoalanine and chloro-phenylalanine.

[0015] Another embodiment comprises a polypeptide comprising a modified yeast phenylalanyl-tRNA synthetase (PheRS), said modified synthetase being mutated at amino acid sequence positions selected from the group consisting of amino acid sequence number (i) 412 and 415; (ii) 415 and 418; (iii) 415 and 437; (iv) 412, 415 and 437; (v) 415, 418 and 437; (vi) 412, 415 and 418; and (vii) 412, 415, 418 and 437 of the polypeptide encoded by SEQ ID NO: 3.

[0016] Some embodiments include a translation system comprising a polynucleotide encoding a modified yeast phenylalanyl-tRNA synthetase, said modified polynucleotide synthetase being mutated at one or more codons encoding an amino acid at a sequence position selected from the group consisting of amino acid sequence position numbers (and ) 412 and 415; (ii) 415 and 418; (iii) 415 and 437; (iv) 412, 415 and 437; (v) 415, 418 and 437; (vi) 412, 415 and 418; and (vii) 412, 415, 418 and 437 of the polypeptide encoded by SEQ ID NO: 3 and wherein said modified synthetase is capable of attaching an unnatural amino acid to a tRNA molecule. In at least one embodiment, the system comprises a host cell. In at least one embodiment, the modified aminoacyl tRNA synthetase is from an organism other than the host cell. In another embodiment, the translation system further comprises a polynucleotide encoding a modified tRNA molecule.

[0017] In certain embodiments, the modified tRNA molecule is from an organism other than the host cell. In certain embodiments, the modified tRNA molecule is from a eukaryotic cell and the host cell is a prokaryotic cell. In yet other embodiments, the cell is an auxotrope.

[0018] In some embodiments, the translation system further comprises a culture medium containing one or more unnatural amino acids. In yet other embodiments, said one or more unnatural amino acids are selected from the group consisting of: azidororleucine, 3- (1-naphthyl) alanine, 3- (2-naphthylalanine, 7-ethynyl-phenylalanine, p-propargyl-oxy-phenylalanine, w-ethynylphenylalanine, 6-ethynyl-tryptophan, 5-ethynyl-tryptophan, acid (R ) -2-amino-3- (4-ethynyl-1H-pyrrol-3-yl) propane, 77-bromophenylalanine, 77-idiophenylalanine, p-azidophenylalanine, 3- (6-chloroindolyl) alanine, 3- (6-bromoindolyl) alanine, 3- (5-bromoindolyl) alanine, azidohomoalanine and 77-chlorophenylalanine.

[0019] Other embodiments relate to a method of introducing an unnatural amino acid into a target polypeptide at one or more specified positions, the method comprising the steps of: providing a translation system comprising at least one unnatural amino acid; providing to the translation system one or more modified yeast phenylalanyl tRNA synthetases as defined above, providing to the translation system a polynucleotide encoding the target polypeptide of interest, and, enabling translation of the target polypeptide of interest, thereby introducing at least one unnatural amino acid into the target polypeptide, with the steps being carried out in any order.

[0020] In certain embodiments, said translation system comprises a cell. In some embodiments, the unnatural amino acid is provided by contacting said translation system with a solution containing the unnatural amino acid. In at least one embodiment, the specificity constant (k_caT / K_M) to activate said unnatural amino acid by said modified AARS is at least 5 times greater than that for said natural amino acid. In certain embodiments, the modified AARS mistakenly attaches no more than 1%, 2%, 3%, 4%, 5%, 6%, 7% or 8% to the tRNA. In yet other embodiments, the tRNA is a modified tRNA. In certain embodiments, said first polynucleotide or said second polynucleotide further comprises either a constitutively active or inducible promoter sequence that controls the expression of tRNA or PheRS. In at least one embodiment, the method further comprises the step of screening cells containing the modified PheRS. In another embodiment, the method further comprises the step of checking for the introduction of an unnatural amino acid. Still other embodiments include a polypeptide produced by the disclosed method.

[0021] Certain embodiments disclosed herein include a method of introducing at least one unnatural amino acid into a target polypeptide at one or more specified locations, the method comprising providing a translation system comprising at least one unnatural amino acid; providing one or more modified PheRS to the translation system; providing to the translation system a polynucleotide encoding a target polypeptide

-6 of interest; and enabling translation of interest, thus introducing at least one unnatural amino acid in the target polypeptide. Some embodiments include a polypeptide produced by this method.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS [0022]

Ligura 1 shows sequence alignment of PheRS variants from conserved sequences of Thermus thermophilus, Escherichia coli and Saccharomyces cerevisiae.

Figure 2 shows several examples of amino acids (naturally occurring or unnatural) used for some of the embodiments disclosed herein.

Figure 3 shows the amino acid sequence for an exemplary polypeptide used in some of the embodiments disclosed herein, dihydrofolate reductase (DHFR). Four proteolytic peptide fragments (designated Peptide A, Peptide B, Peptide C and D peptide) were taken for MALDI and Liquid Spectrum Chromatography / Mass Spectrum (LC-MS / MS) analyzes as underlined.

Figure 4 shows MALDI-MS proteolytic peptide fragments derived from mDHFR expressed in medium supplemented with (a) amino acid 1 (3 mM); (b) amino acid 7 (3 mM) and 1 (0.03 mM); (c) amino acid 2 (3 mM) and 1 (0.03); (d) amino acids 2 (3 mM) and 1 (0.03mM). Tryptophan was not supplemented during induction, except that ImM tryptophan was supplemented in media in (c). Peptide B, containing one Phe codon, is a control. Peptide A contains the amber (Z) codon, an amino acid for which it is assigned based on mass units for Peptide A under various expression conditions. Due to the introduction of lysine with Peptide A (c), C-terminal lysine was cleaved to produce the shorter Peptide A (NGDLPWPPLRNEK) (SEQ ID NO: 4).

Figure 5 Tandem mass spectrum of Peptide A (NGDLPWPPLRNEZK) (SEQ ID NO: 5) derived from DHFR expressed in medium supplemented with amino acid 7 (3mM) and amino acid 1 (0.03 mM). The partial PWPPLRNE sequence (SEQ ID NO: 6) and residue Z (corresponding to amino acid 7) of Peptide A can be assigned from the recorded series of ions y and b, respectively.

Figure 6 shows MALDI-MS proteolytic peptide fragments derived from mDHFR expressed in medium supplemented with (a) amino acid 2 (3 mM) and amino acid 6 (3 mM) and amino acid 1 (0.03 mM) and amino acid 13 (0.2 mM); (b) amino acid 2 (3 mM) and amino acid 6 (0.01 mM) and amino acid 1 (0.03 mM) and amino acid 13 (0.2 mM); (c) amino acid 3

-7 (3 mM) and amino acid 6 (0.01 mM) and amino acid 1 (0.03 mM) and amino acid 13 (0.01 mM). C-peptide, with one Phe codon, is the control.

Figure 7 shows the results of MALDI-MS proteolytic peptide fragments derived from mDHFR expressed in medium supplemented with (a) amino acid 9 (3 mM) and amino acid 6 (0.03 mM) and amino acid 1 (0.01 mM); (b) amino acid 10 (3 mM) and amino acid 6 (0.03 mM) and amino acid 1 (0.03 mM); (c) amino acid 11 (3 mM) and amino acid 6 (0.01 mM) and amino acid 1 (0.03 mM). Peptide D was the control.

Figure 8 shows aminoacylation of yeast tRNA^phecuA (square) and tRNA^phecuA_uG (circle) with lysine using eLysS.

Figure 9 shows LC-MS chromatograms of tryptic digestion of mDHFR polypeptides

Figure 10 shows ATP-PPi exchange rates for phenylalanine, tryptophan and pbromophenylalanine using wild type yeast PheRS and external mutant yeast PheRS T415G or external mutant yeast PheRS T415A.

Figure 11 shows aminoacylation of phenylalanine and tryptophan using wild-type aminoacyl tRNA synthetase or external mutant tRNA synthetase (T415G or T415A).

Figure 12 shows the introduction of lysine (open square), tryptophan (hatched) or p-bromophenylalanine (checkerboard).

Figure 13 shows mass spectra of p-ethynylphenylalanine introduced into a polypeptide using modified tRNA synthetase (T415G) and tRNA^phe with an amber suppressor in a host cell.

Figure 14 shows FACS for the introduction of green fluorescent protein (GFP) amino acids using the amber suppression codon in the protein tested.

Figure 15 shows an example mapping of the aminoacyl tRNA (T415G) mutant plasmid.

Figure 16 shows exemplary mutations introduced into the yeast phenylalanine tRNA synthetase.

DETAILED DESCRIPTION OF THE INVENTION [0023] Proteins occur at the interface of virtually any biological process, from photosynthesis and vision to signal transduction and immune response. Modifying proteins or polypeptides to contain unnatural amino acids has great potential to be used in human therapy, agriculture, biofuels and other areas.

[0024] Aminoacyl-tRNA synthetases catalyze aminoacylation reactions to introduce amino acids into proteins using the appropriate RNA transfer molecules.

Precise manipulation of synthetase activity may alter the aminoacylation specificity for stable attachment of non-canonical amino acids to the intended tRNA. Amino acid analogs can then be introduced into the growing polypeptide chain by the anti-codon-codon interaction between the information RNA (mRNA) and tRNA. Therefore, the introduction of unnatural amino acids into proteins is based on the manipulation of the amino acid specificity of the aminoacyl tRNA synthetase (AARS).

[0025] Aminoacyl-tRNA synthetases function to convert the genetic sequence of the code into biologically functional proteins by a two-step aminoacylation reaction. As an initial step, the related amino acid is activated by AARS in the presence of ATP to form the amino acid adenylate; then AARS catalyzes the esterification reaction to attach the amino acid to the 2'- or 3'-OH terminal ribonucleotide of its related tRNA. After the aminoacylation reaction, the amino acid is directed to the growing polypeptide chain through the loaded tRNA.

[0026] Some embodiments disclosed herein relate to mutated or modified PheRS molecules that have been mutated or modified in such a way that these enzymes are able to attach to the tRNA molecule a replacement amino acid, preferably an unnatural amino acid due to interference between synthetase and the corresponding natural amino acid.

[0027] For example, the interference may be due to interference with Watson-Crick base pairing, interference with varying base pairing, or the creation of a new swinging or different base pairing.

[0028] Certain embodiments relate to a polynucleotide encoding a mutated or modified tRNA or tRNA for a natural amino acid, wherein the natural amino acid is encoded by one or more degenerate wobble codons, the modified tRNA contains a modified anti-codone sequence that creates Watson-Crick base pairing with one of the degenerate wobble codon (s). Preferably, the modified tRNA is not or only slightly charged by the endogenous aminoacyl-tRNA synthetase (AARS) for the natural amino acid. In one embodiment, multiple modified PheRS molecules can be used with one or more tRNA molecules. In one embodiment, one or more modified or mutated PheRS molecules can be used with one or more native tRNA molecules, while in another embodiment the modified or mutated PheRS molecules can be used with the modified or mutated tRNA molecule. In some embodiments, one or more pairs of modified PheRS / tRNA molecules can be used. In some embodiments, heterologous pairs can be used. In certain embodiments, one or more modified or mutated PheRS and / or tRNA may be from the same or different organisms.

[0029] In some exemplary embodiments, a specific PheRS may employ several methods for introducing a replacement amino acid (including an amino acid

Unnatural) to a polypeptide or protein. For example, a single PheRS may use a nonsense codon (such as the amber codon) to introduce a replacement amino acid (such as an unnatural amino acid) at a specific location in the polypeptide. Additionally or instead, a wobble codon (such as UUU) can be used to introduce a replacement amino acid in place of the wobble codon.

[0030] In other exemplary embodiments, many replacement amino acids (such as two different unnatural amino acids) can be introduced into a polypeptide or protein by using different methods. For example, one unnatural amino acid can be introduced at the wobble site, while another unnatural amino acid can be introduced at the amber stop codon. In some exemplary embodiments, the introduction of multiple replacement amino acids (including unnatural amino acids) includes the use of modified PheRS to introduce a variety of different phenylalanine analogues, such as bromophenylalanine and / or p-iodophenylalanine, in the same polypeptide or protein.

[0031] A similar approach is to use a heterologous synthetase and a tRNA initiator mutant from the same organism or a related organism as the tRNA molecule. (See, for example, Kowal, et al., PNAS, 98, 2268 (2001)).

[0032] In some embodiments, the modified or mutated RS interacts with the desired replacement amino acid (whether it is a naturally occurring or non-naturally occurring amino acid) with altered binding specificity and / or an altered catalytic event of the enzyme to replace the amino acid compared to an RS type enzyme wild or wild type appropriate amino acid.

[0033] In enzyme kinetics, k_cat is the first order rate constant corresponding to the slowest stage or stages in the overall catalytic pathway. k_cat represents the maximum number of substrate molecules that can be converted into a product per enzyme molecule per unit of time (which occurs when the enzyme is "saturated" with the substrate) and is therefore often referred to as the number of catalytic cycles (turnover number). K_m is an apparent dissociation constant and is associated with the affinity of the enzyme for the substrate; it is the product of all equilibrium and dissociation constants before the first irreversible step in the pathway. Often, it is a close measure of the enzyme-substrate dissociation constant. k_cat/ K_mis a second order rate constant that refers to free enzyme (and not the enzyme-substrate complex) and is also a measure of the overall efficiency in enzyme catalysis and is also referred to as a specificity constant.

[0034] In certain embodiments, the outer mutant synthetase has improved or improved enzymatic properties, e.g. K_m is greater or smaller, k_catis greater or smaller, k value_caT / K_m is greater or less or similar for an unnatural amino acid relative to a naturally occurring amino acid, e.g. one of the 20 known amino acids. Km of the mutated or modified AARS is preferably equal to or less for the unnatural amino acid than for the corresponding natural wild type amino acid.

[0035] In certain embodiments, the values ​​of k_caT / K_m RS variant can be from 3 times, 5 times, 10 times, 25 times, 50 times, 100 times, 150 times, 200 times, 250 times, 300 times, 350 times, 385 times , 400 times higher than the naturally occurring amino acid.

[0036] In certain embodiments, the modified tRNA interacts with a degenerated wobble codon with an affinity at 37 ° C of at least about 1.0 kcal / mol, 1.5 kcal / mol, 2.0 kcal / mol, 2.5 kcal / mol, 3.0 kcal / mol, 3.5 kcal / mol, 4.0 kcal / mol, 4.5 kcal / mol, 5.0 kcal / mol or more (or any value between them) preferably than the interaction between the unmodified version and the degenerate wobble codon.

[0037] For example, phenylalanine (Phe) is encoded by two codons, UUC and UUU. Both codons are read by a single tRNA which is equipped with a GAA anti-codon sequence. The UUC codon is therefore recognized by standard Watson-Crick pairing between the codon and the anti-codon; UUU is read by a fluctuating pair of GU bases in the first position of the anti-codone (Crick, J. Mol. Biol. 19: 548,1966; Soli and RajBhandary, J. Mol. Biol. 29: 113, 1967). Thermal denaturation of RNA duplexes gave Gibbs free energy estimates for melting GU, GC, AU and AC base pairs as 4.1, 6.5, 6.3 and 2.6 kcal / mol, respectively, at 37 ° C. Thus, the hesitating base pair, GU, is less stable than the Watson-Crick, AU base pair. Modified tRNA^pheequipped with AAA anti-codon (tRNA^pheAAA) was designed by genetic engineering to read the UUU codon and was designed to read these codons faster than the wild-type tRNA^pheGAA [0038] In some embodiments, the RS binding pocket is modified such that the modified RS prefers an unnatural amino acid over the corresponding naturally occurring amino acid. In preferred embodiments, the RS is modified in one or more codons necessary for structural contact between the RS and the amino acid attached to the tRNA. In certain embodiments, one or more codons selected for mutation or modification are selected by computer modeling. While each RS can be modified, the present invention relates to phenylalanyl-tRNA synthetase (PheRS). The modified RS is derived from Saccharomyces cerevisiae, or another yeast cell. In certain embodiments, where RS is PheRS, the enzyme has one or more of the point mutations defined above, in particular the point (N412G), (T415G), (T415A), (S418C) or (S437F) alpha subunit of the enzyme. Point mutations (e.g., the T to G or A mutation at position 415, or the S to C mutation at position 418, or the N to G mutation at position 412, or the S to F mutation at position 437) are located in the aminoacyl synthetase binding pocket region -tRNA (RS).

[0039] In some exemplary embodiments, typical Km values ​​for various analogs with AARS may range from about 15 microM, 20 microM, 30 microM, 50 microM, 75 microM, 100 microM, 150 microM, 200 microM, 300 microM, 400 microM, 440 microM, 500 microM, 1000 microM, 1500 microM, 2000 microM, 3000 microM, 4000 microM, 5000 microM, 6000 microM, or more or any value between them.

[0040] Similarly, the values ​​of k_cat mutant PheRS is preferably equal to or higher for the amino acid analog than for the natural amino acid. For example, k values_cat for different analogues corresponding to PheRS may range from about 0.002 s'¹, 0.0018 s'¹, 0.0015 s'¹, 0.014 s'¹, 0.1 s'¹, 0.3 s'¹, 1 s'¹, 3 s'¹, 5 s'¹, 8 s'¹, 10 s'¹, 13.3 s'¹, 15 s'¹ or more.

[0041] Therefore k_cat/ K_m mutant PheRS is optimally equal to or higher for the amino acid analog than for the wild type natural amino acid. Typical kcat / K values_m may range from about .0001 M '¹ s "¹, .0003 M '¹ s'¹, .005 M '¹ s'¹, .05 M '¹ s'¹, .5 M '¹ s'¹, .547 M '¹ s'¹, 1 M '¹ s'¹, 5 M '¹ s'¹, 10 M '¹ s'¹, 20 M '¹ s'¹, 30 M '¹ s'¹, 32M '¹ s'¹, 500 M ^ s'¹, 600 M''s · ', 1000 M ^ s'¹, 5000 M ^ s'¹, 11000 M ^ s'¹.

[0042] In certain embodiments, PheRS point mutations can be changed to any amino acid, depending on the characteristics of the unnatural amino acid desired for incorporation into the protein / polypeptide of interest. In certain embodiments, an amino acid in a PheRS binding pocket can be mutated to a codon for a small side chain amino acid, aliphatic side chain amino acid, cyclic amino acid, sulfur or hydroxyl group amino acid side chain, aromatic amino acid, basic amino acid, or acidic amino acid (or amide). The choice of amino acid to mutate PheRS at a particular point is routine, depending on the desired result and the desired unnatural amino acid to be included in the test or target polypeptide / protein. For example, if the goal is to enlarge the binding pocket of the PheRS molecule, then an amino acid with an aliphatic side chain or small side chain may be selected for the PheRS mutation. In other cases, if a binding pocket that has a charged pocket is required, then an alkaline or acidic amino acid can be selected for the PheRS point mutation.

[0043] In some embodiments, the modified RS can be used in a translational system, including an auxotrophic host cell or prototropic host cell together with a suppressor tRNA to allow stop codon mapping (such as the nonsense amber, ocher or opal codon, stop codon, which does not occur in a particular organism, any nonsense codon, a codon with four or five base pairs, or any other natural amino acid, which is not present in significant amounts in a protein (such as methionine) to introduce another amino acid, including an amino acid analog. Thus, RS enzymes can be "reprogrammed" for non-specific substrate specificity to facilitate the incorporation of an unnatural amino acid into a polypeptide in a site-specific manner. In specific embodiments, any aromatic unnatural amino acid can be used with the PheRS modification. This reprogramming allows the insertion with high fidelity of an amino acid, including unnatural amino acids, into polypeptides or proteins with or without the use of auxotrophic host cells.

[0044] Reprogramming of the AARS enzyme may include structural or biochemical analysis, including computer modeling or sequence alignment. Since information on the sequence of many AARS molecules is not available, for example in GenBank,

A comparison of sequence alignments is a routine procedure when a given sequence of region of interest is determined.

[0045] The use of auxotrophic host cells may increase the level of incorporation of an unnatural amino acid or reduce the level of misuse of another amino acid instead of the desired unnatural amino acid. For example, after increasing the cellular reactivity of aminoacylation by expression of wild-type PheRS in the host, we have surprisingly found that some of the lazy amino acid analogs can also be introduced into proteins even in the absence of auxotrophic host cells.

[0046] In certain embodiments, the expression of one or more modified or mutated PheRS molecules, one or more modified or mutated tRNA molecules, or both, can be regulated by a constitutive or inducible promoter or other inducible expression system.

[0047] Some embodiments disclosed herein relate to enabling site-specific insertion of one or more unnatural amino acids at any desired position of each protein, (ii) used in both prokaryotic and eukaryotic cells, and allows in vivo testing of mutated proteins in addition to producing larger amounts of purified mutant proteins. In addition, certain embodiments relate to adaptation to introduce any of a wide variety of unnatural amino acids into proteins in vivo. Thus, a variety of site-specific insertions for unnatural amino acids are possible in a particular polypeptide sequence. These types of inserts are optionally all of the same type (e.g., different examples of one type of unnatural amino acid inserted at different places in a polypeptide) or are optionally different types (e.g. different types of unnatural amino acids are inserted at different places in a polypeptide).

[0048] One surprising result disclosed herein shows that the re-construction of the synthetic PheRS enzyme site may develop the ability to introduce replacement amino acids (including unnatural amino acids) into polypeptides or proteins. In some embodiments, compositions and methods for modifying or mutating PheRS may optionally include changing the editing function of the modified or mutated PheRS. In some embodiments, the ability to edit or correct a modified or mutated PheRS is approximately the same as for wild type (unchanged) PheRS. In other embodiments, the ability to edit or correct a modified or mutated PheRS is reduced. In yet other embodiments, the ability to edit or correct a modified or mutated PheRS is eliminated. In some certain embodiments, the PheRS editing or correction function is inseparably associated with modification of PheRS to adjust the replacement amino acid (e.g., modification of the PheRS binding pocket). In other embodiments, changes in the edit or correction function may be performed in addition to modifying the PheRS to adjust the replacement amino acid. In yet other embodiments, the PheRS editing or correction function is

-13niezmieniona. Thus, in addition to modifying or changing the PheRS binding pocket, the domain of correction or editing of the modified PheRS can also be changed to allow increasing the aminoacylation specificity of the replacement amino acid (including the unnatural amino acid) to tRNA (either endogenous or mutated external tRNA), whereas possible hydrolysis adenylate wild type amino acid (s) that may form, results in greater fidelity or specificity for introducing a replacement amino acid (including an unnatural amino acid) into the polypeptide or protein.

[0049] In some embodiments, the rates of introduction of an unnatural amino acid were approximately 65% ​​or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more using modified RS.

[0050] As disclosed herein, inter alia, the exact RS crystal structure to be modified or the homologous RS crystal structure can be used to model the enzyme-amino acid molecular interaction to determine contact points between RS and the corresponding naturally occurring amino acid and / or contact points between RS and selected an unnatural amino acid that is desirable for incorporation into a polypeptide. For example, in certain embodiments provided herein, the crystal structure of PheRS Thermus thermophilus complexed with phenylalanine was used to model the molecular design of PheRS Saccharomyces cerevisiae, due to sequence identity of about 40% in the region of the synthetase active site. The threonine mutation at position 415 to Glycine or Alanine (T415G or T415A, respectively) increased the active site and enabled the placement of larger phenylalanine analogues. Mutations such as the one that interrupts the pairing of Watson-Crick bases with the naturally occurring amino acid designated by each RS allow for increased specificity for the introduction of an unnatural amino acid and to reduce the mis-entry of another amino acid base. As shown in the Examples and Figures below, (T415A) phenylalanine yeast aminoacyl tRNA (PheRS) revealed a 5-fold preference for bromophenylalanine than for naturally occurring phenylalanine. Thus, it is possible to change the aminoacyl tRNA (RS) synthetase molecule to preferentially introduce the desired amino acid with 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x , 10 times or more, depending on the properties of each RS and the desired amino acid (including unnatural amino acid).

[0051] In one particular embodiment, it has been found that the space around the para position of the aryl ring bound to Phe can be slightly reduced to exclude other aromatic amino acids such as Trp, while at the same time placing a non-naturally occurring amino acid such as pBrF. Thus, one embodiment discloses introducing the aryl bromide functional group into the polypeptide at a programmed position by providing chemoselective ligation via palladium catalyzed cross-coupling with ethylene or acetylene reaction partners. While such

Reprogrammed or modified RS can be used in any number of host cells, including auxotrophic host cells, a high level of introduction of the desired amino acid (or analog) of the modified RS of certain embodiments, and high protein production yields, making use of auxotrophic host cells unnecessary.

[0052] Since the active site region for almost all amino acid synthetases is known and easily deduced, such an exemplary technique can be used for other AARS to reprogram the amino acid specificity from a naturally occurring amino acid to an unnatural amino acid with the expectation of success and without undue experimentation.

[0053] As an illustrative example, threonine at position 415 in yeast PheRS is equivalent to threonine 251 in E. coli PheRS. Thus, mutations in yeast PheRS (T415G) allow the activation of many Phe analogues. (See Examples here.) In addition, point mutations and / or the use of an auxotrophic host cell have resulted in reduced base error in the yeast variant PheRS T415G.

[0054] In another particular embodiment disclosed herein, mutated yeast transfer RNA (ytRNA^phecuA), whose pairing of Watson-Crick bases between amino acid position 30 and amino acid position 40 was disturbed, loaded with p-bromo-phenylalanine (pBrF) by co-expressed yeast phenylalanine. In certain embodiments, the AARS amino acid binding pocket is approximately 200, approximately 100, approximately 75, approximately 50, approximately 25, approximately 10, approximately 5 or less or more amino acids. In some embodiments, the amino acids to be mutated at the active site or binding site are adjacent amino acid segments. In other embodiments, the amino acids to be mutated are placed in close proximity to each other, but not adjacent.

[0055] In some embodiments, the natural amino acid is encoded by two or more genetic codes (and thus encoded by degenerate genetic codes). In most cases, if not all, this includes 18 out of 20 naturally occurring amino acids, excluding Met and Trp. Under these circumstances, to recognize all degenerate genetic codes for a natural amino acid, the wild-type tRNA anti-codon loop relies on both fluctuating base pairing and pure Watson-Crick base pairing. The subject modified tRNA contains at least one modification in the anti-codon loop such that the modified anti-codon loop creates a pairing of Watson-Crick bases to one of the degenerate genetic codes that the tRNA previously only bound by fluctuating base pairing.

[0056] Because Watson-Crick base pairing is invariably stronger and more stable than fluctuating base pairing, the modified tRNAs will preferably bind to previous fluctuating base pairing of the genetic code

-15 (now by pairing Watson-Crick rules) compared to the previous pairing of Watson-Crick rules (now by fluctuating pairing of rules). Thus, an analogue can be introduced in the subject codon if a natural amino acid analogue is attached to the modified tRNA, which may or may not be the same as the natural amino acid encoded by said codon.

[0057] Thus, in some embodiments, if desired, to introduce certain amino acid analogs at the codons for Met or Trp, a tRNA for a natural amino acid (e.g., Met tRNA, Trp tRNA, or even Phe tRNA, etc.) can be modified to recognize the Met or Trp codon. In this kind of unique situation, both modified tRNA and natural tRNA compete to bind the same (single) genetic code by pairing with Watson-Crick principles. Some, but not all, of these codons will accept their natural amino acids, while others may accept amino acid analogs carried by the modified tRNA. Other factors, such as the abundance of a natural amino acid compared to the abundance of an analogue, may affect the final result. (See Examples disclosed below.)

[0058] In certain preferred embodiments, the modified tRNA is not charged or merely insufficiently charged by the endogenous aminoacylotRNA synthetase (AARS) for any natural amino acid such that the modified tRNA significantly (if not exclusively) carries the amino acid analog but not the natural amino acid . Although the subject modified tRNA may still be useful if it can be loaded by endogenous AARS with a natural amino acid.

[0059] In certain embodiments, the modified tRNA with an attached amino acid analog has such an overall shape and size that the analog-tRNA is a ribosomally acceptable complex, i.e., the analog-tRNA complex can be accepted by prokaryotic or eukaryotic ribosomes in the in vivo translation system or in vitro.

[0060] Preferably the modified PheRS specifically or preferentially attaches an analog to the modified tRNA relative to any natural amino acid. In a preferred embodiment, the specificity constant for activating the analog by modified AARS (referred to as kcat / KM) is equal to or greater than at least about 2 times greater than that of the natural amino acid, preferably about 3 times, 4 times, 5- times, 6 times, 7 times, 8 times, 9 times, 10 times or more than that of a natural amino acid.

[0061] In certain embodiments, the modified tRNA further comprises a mutation at the fourth extended anti-codon site for increased translational efficiency.

[0062] The use of extended codons is based on suppressing translation frame shift. Four-base codons have the potential to introduce many unnatural amino acids within the same protein. For example, the UAGA quadruplet can be decoded with tRNA^Leu with UCUA anti-codon with 13 to 26% efficiency. (See, for example, Moore, et al., J. Mol. Biol., 298: 195 (2000)). The use of extended codons alone has potential problems such as frame reading of the first three

-16 bases as the triplet in the extended codon competes with the overall suppression of the frame shift. In some cases, extended codons based on rare codons or nonsense codons can reduce erroneous sense reading and frame shift suppression at other undesirable locations. These problems can be solved, however, using extended codon / anti-codon and modified AARS and / or tR NA as indicated in some of the embodiments disclosed herein.

[0063] Thus, to summarize, specific codons are reserved for use in the methods disclosed herein using a mutated or modified PheRS and / or modified or mutated tRNA to introduce a replacement amino acid (including a naturally occurring or unnatural amino acid). Such methods may include the use of amber decoding (ocher, umber or other suppressor tRNA) that reads stop codons (TAGs), preferential decoding (ang. bias decoding), which uses unused tRNAs responsible for codon preferences (codon bias), wobble decoding that creates tRNAs that read wobble codons and extended (4-5 bases or more) codons that use "suppressor" tRNAs that use 4 base or 5 base (or more) anti-codones.

[0064] At least one other embodiment provides a method of introducing an amino acid analog into a target protein at one or more specified positions, the method comprising: (1) providing to the environment a first subject polynucleotide for a modified tRNA, or a subject modified tRNA; (2) providing to the environment a second subject nucleotide encoding the modified PheRS, wherein the modified PheRS is capable of loading the modified tRNA with an analogue; (3) providing an analogue to the environment; (4) providing a polynucleotide matrix encoding the target protein, wherein the codon on the polynucleotide matrix for the specific position only forms a pairing of Watson-Crick bases with the modified tRNA; and, (5) enabling translation of the polynucleotide matrix to occur, thereby introducing the analog to the target protein at a particular position, wherein steps (1) - (4) are performed in any order.

[0065] In certain embodiments, the method further includes verifying the incorporation of the analog by, for example, mass spectrometry, protein sequencing, amino acid tagging, e.g., by fluorescence, radioactivity, etc., ELISA or other antibody screening tests, functional screening tests or tests or other methods .

[0066] In some embodiments, the method introduces the analog in position with a yield of at least about 50% or 60%, 70%, 80%, 90%, 95%, 99% or nearly 100%.

Definitions [0067] Before proceeding with the detailed description of certain embodiments, it should be understood that the invention is not limited to specific biological compositions or systems, which may, of course, be different. It should also be understood that the terminology used herein is for the purpose of describing particular examples only

-17 performances and is not intended to be limiting. The singular forms used in this description and the appended claims include their references to the plural, unless the content clearly indicates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules and the like.

[0068] Unless specifically defined below, the terms used herein generally have their common meaning in the art, in the general context of the present invention, and in the specific context in which the term is used. Some terms are discussed below or elsewhere in the description to provide additional guidance to the physician for a description of the composition and methods of the invention as well as for implementation and use. The scope and meaning of each term used will be apparent from the particular context in which the term is used.

[0069] "Approximately" or "approximately" means the generally acceptable error rate for a measured quantity given the nature and precision of the measurements. Typical examples of error rates are within 20 percent (%), preferably 10%, and more preferably within 5% of the value or range of values ​​given. Alternatively, particularly in biological systems, the terms "about" and "approximately" may mean values ​​in the order of magnitude, preferably in the 5-fold range, and more preferably in the 2-fold range of the given value. The numerical amounts given here are approximate unless otherwise stated, which means that the word "approximately" or "approximately" can be inferred when not explicitly indicated.

[0070] "Amino acid analog", "non-canonical amino acid" or "non-natural amino acid", "unnatural amino acid", "unnatural amino acid" and the like can be used interchangeably and are intended to include all amino acid-like compounds that are similar in structure and / or the general shape of one or more of the twenty L-amino acids commonly found in naturally occurring proteins (Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gin or Q, Arg or R, Ser or S, Thr or T, Val or V, Trp or W, Tyr or Y as defined and listed in WIPO Standard ST.25 (1998), Appendix 2, Table 3). The amino acid analog may be natural amino acids with modified side chains or skeletons. Amino acids may also be naturally occurring D-amino acids instead of the L form. Preferably, these analogs are usually not "substrates" for aminoacyl tRNA synthetases (AARS) due to the typically high specificity of AARS. Although occasionally, certain analogues with structures or shapes close enough to natural amino acids may be mistakenly introduced into proteins using AARS, especially modified AARS with relaxing substrate specificity. In a preferred embodiment, the analogs share skeletal structures and / or even most of the side chain structure of one or more natural amino acids, with the only difference (s) that they contain one or more modified groups in the molecule. Such modification may include, without limitation, substitution of an atom (like N) with a related atom (like S), addition of groups (like methyl or hydroxyl, etc.) or atom (like Cl or Br, etc.), removal of a group (supra) , covalent bond substitution (single bond

-18 double bond, etc.) or combinations thereof. Amino acid analogs may include ahydroxy acids and β-amino acids and may also be referred to as "modified amino acids" or "unnatural AARS substrates".

[0071] Amino acid analogues can be either naturally occurring or non-naturally occurring (e.g. synthesized). As will be appreciated by those skilled in the art, any structure for which a set of rotamers is known or can be generated can be used as an amino acid analogue. The side chains may be of the (R) or (S) (or D or L configuration). In a preferred embodiment, the amino acids have the (S) or L configuration.

[0072] Preferably, the overall shape and size of the amino acid analogs are such that when attached to (natural or modified or re-designed) tRNA using (natural or re-designed) AARS, the analog-tRNA is a ribosomally acceptable complex, i.e. a tRNA complex -analog can be taken by prokaryotic or eukaryotic ribosomes in the in vivo or in vitro translation system.

[0073] "Anchor residues" are residue positions in AARS that maintain critical interactions between AARS and the natural amino acid backbone.

[0074] A "skeleton" or "matrix" includes skeleton atoms and any stable side chains (such as side chain anchor moieties) of a protein (e.g., AARS). For calculation purposes, the analog skeleton is treated as part of the AARS skeleton.

[0075] "Skeletal structure of a protein" or grammatical equivalents used herein means three-dimensional coordinates that define the three-dimensional structure of a particular protein. Structures that contain the skeletal structure of a protein (naturally occurring protein) are nitrogen, carbonyl carbon, α-carbon, and carbonyl oxygen, along with the vector direction from α-carbon to β-carbon.

[0076] The skeletal structure of a protein, which is entered into a computer for computational molecular structural prediction or interaction, can either contain the coordinates of both the main chain and the side chains of the amino acids or only the skeleton, i.e. with the removed coordinates for the side chains of amino acids. If this is done first, the atoms in the side chain of each amino acid protein structure can be "stripped" or removed from the protein structure, as is known in the art, leaving only the coordinates for "skeletal" atoms (nitrogen, carbon and carbonyl oxygen and α-carbon , and hydrogen atoms attached to the nitrogen and α-carbon).

[0077] Optionally, the skeletal structure of the protein may be modified prior to the analysis described below. In this embodiment, the representation of the initial skeletal structure of the protein is reduced to a description of the spatial distribution of its secondary structure elements. The relative positions of the secondary structure elements are defined by a set of parameters called super secondary structure parameters. These parameters are assigned values ​​that can be systematically or randomly changed to change the arrangement of the secondary elements of the structure to introduce pronounced flexibility of the skeleton. Then the coordinates are changed

-19atom skeleton to account for altered super-secondary structural parameters and such new coordinates are introduced into the system for use in the next automated protein design. For more information, see Pat. No. US 6,269,312, the entire content of which is incorporated herein by reference.

[0078] "Conformational energy" generally refers to energy associated with a given "conformation" or three-dimensional structure, macromolecules such as energy associated with the conformation of a particular protein. Interactions that tend to stabilize proteins have energies that are shown to be negative, whereas interactions energy values ​​that destabilize proteins have positive energy values. Thus, conformational energy for any stable protein is quantitatively represented by negative conformational energy. In general, conformational energy for a given protein will be related to protein stability. In particular, molecules that have lower (or more negative) conformational energy are usually more stable, e.g., at higher temperatures (i.e., have greater "thermal stability"). Accordingly, the conformational energy of a protein can also be referred to as "stabilization energy".

[0079] Typically, conformational energy is calculated using the "force field" energy, which calculates or estimates energy supply from various interactions dependent on the conformation of the molecule. The force field consists of conditions that include the conformational energy of the alpha carbon skeleton, side chain - skeleton interactions, and side chain - side chain interactions. Typically, backbone or side chain interactions include conditions for binding rotation, binding twisting, and binding length. Skeleton-side-chain and side-chain-side interactions include van der Waals interactions, hydrogen bonds, electrostatic interactions and solvation conditions. Electrostatic interactions may include Coulomb interactions, dipole interactions and quadrapole interactions). Other similar terms may also be included. Force fields that can be used to determine the conformational energy of a polymer are well known in the art and include, to name a few, force fields CHARMM (see, Brooks et al., J. Comp. Chem. 1983.4: 187-217; MacKerell et al., in The Encyclopedia of Computational Chemistry, Vol. 1: 271-277, John Wiley & Sons, Chichester, 1998), AMBER (see, Comell et al., J. Amer. Chem. Soc. 1995, 117: 5179; Woods et al., J. Phys. Chem. 1995, 99: 3832-3846; Weiner et al., J. Comp. Chem. 1986, 7: 230; and Weiner et al., J. Amer. Chem. Soc. 1984, 106: 765) and DREIDING (Mayo et al., J. Phys. Chem. 1990, 94-: 8897).

[0080] In a preferred embodiment, hydrogen bonding and electrostatic conditions are as described in Dahiyat & Mayo, (Science 1997 278: 82). The force field can also be determined to contain atomic conformational conditions (bond angles, bond lengths, torsion) as in other references. See, e.g., Nielsen, et al., Prot. Eng., 12: 657662 (1999); Stikoff, et al., Biophys. J., 67: 2251-2260 (1994); Hendsch, et al., Prot. Sci., 3: 211-226 (1994); Schneider, et al., J. Am. Chem. Soc., 119: 5742-5743 (1997); Sidelar, et al., Prot. Sci., 7: 1898-1914 (1998). Solvation conditions may be included. See, e.g., Jackson, et al., Biochemistry, 32: 11259-11269 (1993); Eisenberg, et al., Nature, 319: 199-203 (1986); Street AG and Mayo SL;

-20 Folding & Design, 3: 253-258 (1998); Eisenberg and Wesson, Prot. Sci., 1: 227-235 (1992); Gordon & Mayo, supra.

[0081] "Coupled residues" are residues in a molecule that interact through any mechanism. The interaction between two residues is therefore hereinafter referred to as "coupling interaction". Conjugated residues generally contribute to polymer suitability by coupling interaction. Typically, the coupling interaction is a physical or chemical interaction such as electrostatic interaction, van der Waals interaction, hydrogen bonding interaction or a combination thereof. Consequently, the coupling interaction, by changing the identity of each residue, will affect the "usefulness" of the molecule especially if the change interferes with the coupling interaction between the two residues. The coupling interaction can also be described by the parameter of the distance between residues in the molecule. If the residues are at a certain limit distance, they are considered to be interacting.

[0082] "Fitness" is used to determine the level and extent to which a particular property or combination of properties in particular in a molecule, for example, protein, is optimal. In certain embodiments of the invention, the usefulness of the protein is preferably determined by the properties that the user wants to improve. Thus, for example, the utility of a protein may refer to protein thermal stability, catalytic activity, affinity, solubility (e.g. in an aqueous or organic solvent) and the like. Other examples of useful properties include enantioselectivity, activity against unnatural substrates, and alternative catalytic mechanisms. Coupling interactions can be modeled as a means of assessing or predicting suitability (stability). Suitability can be determined or evaluated experimentally or theoretically, for example, computationally.

[0083] Preferably, the suitability is quantified such that each molecule, eg, each amino acid, will have a particular 'value of usefulness'. For example, the utility of a protein may be the rate at which the protein catalyzes a specific chemical reaction, or the ligand binding affinity of the protein. In a particularly preferred embodiment, the utility of the protein refers to the conformational energy of the polymer and is calculated, e.g., by any method known in the art. See e.g. Brooks, et al., J. Comp. Chem., 4: 187-217 (1983); Mayo, et al., J. Phys. Chem., 94: 8897-8909 (1990); Pabo, et al., Biochemistry, 25: 5987-5991 (1986), Lazar, et al., Prot. Sci., 6: 1167-1178 (1997); Lee, et al., Nature, 352: 448-451 (1991); Colombo, et al., J. Am. Chem. Soc., 121: 6895-6903 (1999); Weiner, et al., J. Am. Chem. Soc., 106: 765-784 (1984): In general, the usefulness of a protein is quantified such that the usefulness value increases when a property or combination of properties is optimized. For example, in embodiments where the stability of the protein is optimized ((conformational) energy preferably decreases), the value of suitability may be negative conformational energy; i.e. F = -E.

[0084] "Contribution to suitability" of a protein residue refers to the level or range of f (i_and) in which the rest and_and, having identity a, contributes to the total usefulness of the protein. Thus, for example, if a change or mutation of a particular amino acid residue significantly decreases

-21 protein suitability, it is said that the remainder has a high contribution to the suitability of the polymer. Conversely, usually some rests and_and in a protein they can have many possible identities without affecting the usefulness of the protein. These residues therefore have a low contribution to the usefulness of the protein.

"Dead-end elimination" (DEE) is a deterministic search algorithm that tries to systematically eliminate bad rotamers and combinations of rotamers until only one solution remains. For example, amino acid residues can be modeled as rotamers that interact with a solid backbone. The theoretical basis for DEE ensures that, if the search for DEE coincides, the solution is the global minimum conformational energy (GMEC) without uncertainty (Desmet et al., 1992).

[0085] The elimination of dead ends is based on the following concept. Consider two rotamers, and_r and and_t, at residue i, and a set of all other rotamer configurations {S} at all residues except i (whose rotamer j_s is an element). If the energy between pairs contributes between and_r and j_s is greater than the energy between t and j pairs_sfor all {S}, then rotamer and_r cannot occur in the global minimum of conformational energy and can be eliminated. This concept is expressed mathematically by inequality.

+ X and *) * ⁺ Σ { ^S ) (Equation A)

7 ** [0086] If this expression is true, a single rotamer and_r can be eliminated (Desmet et al., 1992).

[0087] In this embodiment, Equation A is not easy to perform computational processing, because to perform the elimination it is required to calculate the entire area of ​​the sequence (rotamer). To simplify the problem, you can use the boundaries implied by Equation A:

E {i_r) + £ min (S) E (/ ,, /_s)> E (i <) + £ roax {s) E (/_f, /,) {S) (Equation B) [0088] Using analogous argumentation, Equation B can be extended to eliminate pairs of non-GMEC-compatible rotamers. This is done by establishing that pairs of rotamers and_rwith the rest of i and j_s with the rest of j, they always contribute to higher energies than rotamers and_at and j_v with all possible combinations of rotamers {L}. Similar to Equation B, the close association with this statement is given by:

h) + Σ ^min(Wr. H,> Źiu, λ) ⁺ Σ niax (t)_C<_Łu, j_v, k!) (Equation C (i »/, 7 where ε is the combined energies for rotamer pairs" £ (ir) + £ (/_s) + £ (w>) (Equation Di.

-22 = Η (4.Λι) + E {jJĄ ((Equation E).

[0089] This leads to a double elimination of rotamer pairs and_r and j_sbut does not completely eliminate individual rotamers because everyone can exist independently in GMEC. This double elimination step reduces the number of possible pairs (reduces S) that must be judged to the right of Equation 6, allowing more individual rotamers to be eliminated.

[0090] The single and double criteria set out by Desmet et al. Do not disclose the specific conditions that lead to the determination of rotamers that are larger dead ends. For example, it is possible that the energy input of rotamer and_t is always lower than and_r without the maximum of it being below the minimum and_r. To solve this problem, Goldstein 1994 introduced a modification of criteria that determine whether the energy profiles of two rotamers cross. If not, higher rotamer energy can be considered a dead end. Double calculation uses much more computational time than single calculation. To accelerate this process, other computational methods have been developed to predict double calculations that will be most efficient (Gordon & Mayo, 1998). These types of modifications, collectively called fast double, significantly improve the speed and efficiency of DEE.

[0091] Several other modifications also increase DEE. Rotamers from many residues can be combined into so-called superrotamers to stimulate further elimination (Desmet et al., 1994; Goldstein, 1994). This has the advantage of eliminating many rotamers in one step. In addition, conformational "separation" of space between rotamers has been shown to improve DEE performance (Pierce et al., 2000). Separation handles the following special case. Consider the rotamer and_r. If the rotamer and_t| contributes to less energy than and_r for part of the conformational space, and rotamer i ^ has less energy than i_r for the remaining faction, then and_r can be eliminated. This case would not be detected by the less sensitive Desmet or Goldstein criteria. In preferred embodiments, as described herein, all of the DEE improvements described have been applied.

[0092] For a further discussion of these methods, see Goldstein, Biophysical Journal 66, 13351340 (1994); Desmet, et al., Nature 356,539-542 (1992); Desmet, et al., The Protein Folding Problem and Tertiary Structure Prediction (Jr 'KM & Grand, S. L' eds.), Pp. 307337 (Birkhauser, Boston, 1994); De Maeyer, et al., Folding & Design 2, 53-66 (1997), Gordon, and Mayo, J. Comp. Chem. 19, 1505-1514 (1998); Pierce, et al., J. Comp. Chem. 21, 999-1009 (2000).

[0093] Another calculation called SCREAM (Side-Chain Rotamer Energy can be used

Analysis Method). SCREAM allows studying the mechanism of discrimination of non-related amino acids by calculating the relative binding energy of 20 natural amino acids to a specific AARS. (See, for example, McClendon, et al., Prot. Eng. Design & Select. 19:

195-203 (2006)).

[0094] In a first step, the rotamer energy spectrum for a single amino acid in the empty skeleton is calculated, without any mobile side chains. Then, starting with the lowest rotamers from the hollow skeleton, fill the side chains, but eliminate collisions. For example, place side chains anywhere, estimating the energies of low excitations from the empty skeleton spectrum, and calculate the interaction in pairs, eliminating collision configurations. So, E_tot (A, B) = E_sey (A) + E_seij (B) + Ei "_t. (A, B) = E_self(A, B) and E_tot (A, B, C) = E_seand j (A) + E_seand j (B) + E_seand / (C) + Ε_ιηΙ (A, B) + E_int (A, C) + E_inl (B, C) = E_seand_f(AB) + E_seand_f(C) + E_int (A, C) + E_int (B, C) = E_seand / (A, B) + E_setf (C) + E_in, (AB, C) establishes a recursive relationship. Then, all low lying side chain excitations must be analyzed until the energy distribution for rotamer energy in the empty skeleton stops increasing. In short, the ground state energy is evaluated for all residues, followed by the set of rotamers with the lowest linear total energy, and finally the next lowest linear total energy, and so on. In addition, electrostatic interactions should be addressed because the charges polarize the environment to protect the charges and reduce the desired amino acid interaction. Since molecular dynamics modeling methods usually do not take into account polarization, the deviation relates to salt bridges. To overcome this, residues are neutralized and parameters re-evaluated according to DREIDING parameterization.

[0095] For DREIDING parameterization, the lost charged-charged and charged-dipole interactions are compensated by entering the term hydrogen bond. This can be done in conjunction with other programs, including CHARMM, as described herein. So, using a crystallographic structure from a specific AARS, or homologous AARS from another organism, we can use a program such as SCREAM, and HierDock, or others, to predict the binding conformation and binding energy of each of the 20 natural amino acids at the binding site in the best binding mode and activation mode, by ordering calculations according to which amino acids compete for binding to a particular AARS.

[0096] In particular, selective binding is the first analysis that provides an amino acid and ATP molecule to bind to an active AARS site. This sometimes causes conformational changes. Then, selective activation of AARS to catalyze the formation of a covalent bond between the amino acid and ATP, forms aminoacyl adenylate complexed with AARS and removes inorganic pyrophosphate. Third, if there is an erroneous activation of a non-related amino acid, AARS can hydrolytically cleave the aminoacyl adenylate complex (as a pre-transfer correction). Finally, if non-related aminoacyl adenylate survived, AARS can hydrolytically cleave the aminoacyl-tRNA complex (post-transfer correction).

[0097] Thus, such computational programs allow reliable predictions of the likelihood of natural amino acids that will complete binding and aminoacylation by wild-type or mutated AARS enzymes. The use of multiple programs (such as SCREAM and HierDock together) reduces the speed of incorrect policy entry and

-24 allows greater predictability in choosing the location of amino acids that specifically bind an amino acid.

[0098] Still other computer modeling programs include SCAP (Side Chain Amino Acid Prediction Program) (Xiang and Honig, J. Mol. Biol. 311, 421-430 (2001)) and SCWRL (Side Chain Replacement With a Rotamer Library) which is useful for adding side chains to a protein skeleton based on a skeleton dependent library of rotamers. The SCWRL library provides lists of chil-chi2-chi3-chi4 values ​​and their relative probabilities for residues in the given phi-psi values, and examines these conformations to minimize side-skeleton and side-chain side-collision collisions. (See, for example, Bower, et al., J. Mol. Biol., 267, 1268-1282 (1997)).

[0099] Computational predictability is the result of a large part of the use of known AARS nucleic acid and / or amino acid sequences. For example, the catalytic domain is preserved for all members of a particular class of AARS enzyme. (O'Donoghue and Luthey-Schulten, Microbiol. And Mol. Biol. Rev .: 550-573 (2003); Diaz-Lazcoz, et al., Mol. Biol. Evol. 15 (11): 1548-1561 (1998 ); Wang, et al., Chem. Commun. 1-11 (2002)).

[0100] "Expression system" means a host cell or cellular components and a compatible vector under appropriate conditions, for example, for the expression of a protein encoded by foreign DNA carried by the vector and introduced into the host cell. Typical expression systems include E. host cells coli and vector plasmids, insect host cells such as Hi5, Sf9 or S2 cells and baculovirus vectors, Drosophila cells (Schneider cells) and expression systems, and mammalian host cells and vectors.

[0101] "Host cell" means any cell of any organism that has been selected, modified, transformed, cultured or used or manipulated in any manner for the cell to produce a substance. The host cell may be auxotrophic, which is not capable of synthesizing at least one particular compound needed for its development and maintenance, and must obtain the compound from another source, such as its environment or culture medium. In addition, auxotrophic host cells may have one, two, three, four or more levels of auxotrophy, so that it is unable to synthesize one, two, three, four or more organic compounds necessary for its growth and maintenance, respectively. For example, a host cell may be one that is manipulated to express a given gene, DNA or RNA sequence, protein or enzyme. Host cells can be cultured in vitro or one or more cells in a non-human animal (e.g., a transgenic animal or transiently transfected animal).

[0102] Certain embodiments disclosed herein expressly only use a cell-free expression or translation system, not a host cell. Some other embodiments clearly only use auxotrophic host cells.

-25 Still some other embodiments expressly use only non-auxotrophic host cells or prototropic host cells.

[0103] Sequence similarity may be relevant to some embodiments because they may include the steps of comparing the sequences with each other, including wild-type sequences to one or more mutants. Such comparisons typically include alignment of polymer sequences, e.g., using programs and / or sequence alignment algorithms that are well known in the art (e.g., to name a few, BLAST, FASTA and MEGALIGN). One of skill in the art can readily recognize that in such matches where the mutation involves the insertion or removal of a residue, the sequence alignment will introduce "gaps" (typically represented by a dash, "-" or "A") into the polymer sequence not containing the inserted or deleted residue.

[0104] "homologous" in all its grammatical forms and spelling variants refers to the relationship between two molecules (for example, protein, tRNA, nucleic acids) that have "evolutionary common origin", including superfamily proteins in the same species of organism and homologous proteins from other species of organisms. Such proteins (and the nucleic acids encoding them) possess sequence and / or structure homology, which reflects their sequence similarity, in terms of percent identity or the presence of specific residues or motifs and preserved positions. Homologous molecules also often have similar or even identical functions.

[0105] In some aspects, homologous may comprise a sequence that is at least 50% homologous, but has a homologous structure in three dimensions, that is, it comprises a substantially similar surface charge or presentation of hydrophobic groups. Because hydrogen bonds, van der Waals forces, and hydrophobic interactions can act to bind an amino acid to the AARS binding pocket, manipulation of the AARS structure can also change one or more of these forces.

[0106] Thus, as used herein, proteins and / or protein sequences are "homologous" when they are derived, naturally or artificially, from a common ancestor of the protein or sequence. Similarly, nucleic acids and / or nucleic acid sequences are homologous when they are derived, naturally or artificially, from a common ancestor of the nucleic acid or nucleic acid sequence. For example, any naturally occurring nucleic acid can be modified by any available mutagenesis method to contain one or more selector codons. When expressed, this mutagenized nucleic acid encodes a polypeptide containing one or more unnatural amino acids. The mutation process can of course additionally change one or more standard codons, thereby also changing one or more standard amino acids in the resulting mutant protein. Homology generally results from sequence similarity between two or more nucleic acids or proteins (or their sequences). The exact percentage of sequence similarity that is useful in determining homology varies with the subject nucleic acid and protein, but only 25% of sequence similarity is routinely

-26 used to determine homology. Higher levels of sequence similarity, e.g. 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more, can also be used to determine homology. Methods for determining percent sequence similarity (e.g., BLASTN and BLASTP using default parameters) are described herein and are widely available.

[0107] The term "sequence similarity" in all its grammatical forms refers to the degree of identity or agreement between nucleic acid or amino acid sequences that may or may not have evolutionary common origin (see, Reeck et al., Supra). However, in common use and the present application, the term "homologous" when modified by an adverb such as "highly" may refer to sequence similarity and may or may not refer to common evolutionary origin.

[0108] A nucleic acid molecule is "capable of hybridizing" to another nucleic acid molecule such as cDNA, genomic DNA or RNA, if the single-stranded form of the nucleic acid molecule can fuse into another nucleic acid molecule under appropriate conditions of temperature and ionic strength of the solution ( see Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Temperature and ionic strength conditions determine the "stringency" of hybridization conditions. Low stringency hybridization conditions corresponding to T can be used to pre-screen homologous nucleic acids_m (melting point) 55 ° C, e.g. 5> <SSC, 0.1% SDS, 0.25% milk and no formamide; or 30% formamide, 5> <SSC, 0.5% SDS). Medium stringent hybridization conditions correspond to a higher T_me.g. 40% formamide, with 5x or 6> <SSC. High stringency hybridization conditions correspond to the highest T_me.g. 50% formamide, 5 * or 6> <SSC. SSC means 0.15M NaCl, 0.015M Na citrate. Hybridization requires that two nucleic acids contain complementary segments of the genetic or amino acid sequence, although depending on the stringency of hybridization conditions, base discrepancies are possible.

[0109] Adequate stringency for hybridization of nucleic acids depending on the length of the nucleic acids and the degree of complementarity of variables is well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the higher the T value_m for nucleic acid hybrids having these sequences. Relative stability (corresponding to a higher T_m) hybridization of nucleic acids decreases in the order: RNA: RNA, DNA: RNA, DNA: DNA. For hybrids larger than 100 nucleotides, the equations for calculating T were derived_m (see Sambrook et al., supra, 9.50-9.51). For hybridization from shorter nucleic acids, i.e. oligonucleotides, mismatch positions are becoming increasingly important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., Supra, 11.7-11.8). The minimum length for a hybridizable nucleic acid is at least about 10 nucleotides; more preferably at least about 15 nucleotides; and more preferably the length is at least about 20 nucleotides.

[0110] Unless otherwise specified, the term "normal hybridization conditions" refers to T_m about 55 ° C, and uses the conditions as given above. In a preferred embodiment, T._m is 60 ° C; in a more preferred embodiment, T_m is 65 ° C. In a specific embodiment, "high stringency" refers to hybridization and / or wash conditions at 68 ° C in 0.2 * SSC, at 42 ° C in 50% formamide, 4xSSC or under conditions that provide levels of equivalent hybridization to those observed in one of these two conditions.

[0111] Suitable hybridization conditions for oligonucleotides (e.g., oligonucleotide probes or primers) are usually slightly different than for full-length nucleic acids (e.g., full-length cDNA), due to the lower melting points of the oligonucleotides. Because the melting point of oligonucleotides depends on the length of the oligonucleotide sequence of pages, the respective hybridization temperatures vary depending on the oligonucleotide molecules used. Exemplary temperatures may be 37 ° C (for 14-base oligonucleotides), 48 ° C (for 17-base oligonucleotides), 55 ° C (for 20-base oligonucleotides) and 60 ° C (for 23-base oligonucleotides). Exemplary suitable hybridization conditions for oligonucleotides include washing in 6 x SSC / 0.05% sodium pyrophosphate or other conditions that provide equivalent levels of hybridization.

[0112] "Polypeptide", "peptide" and "protein" are used interchangeably to designate the chain of amino acids joined together by chemical bonds called "peptide bonds". The protein or polypeptide, including the enzyme, may be "native" or "wild-type", which means that it occurs in nature; or it may be a "mutant", "variant" or "modified" meaning that it has been made, altered, derived or in some other way different or changed with respect to a native protein or other mutant.

[0113] "Rotamer" is defined as the set of possible conformers for each amino acid or analog side chain. See Ponder, et al., Acad. Press Inc. (London) Ltd. pp. 775-791 (1987); Dunbrack, et al., Strucia. Biol. 1 (5): 334-340 (1994); Desmet, et al., Nature 356: 539-542 (1992). A "rotamer library" is a collection of a set of possible / acceptable rotameric conformations for a given set of amino acids or analogs. There are two main types of rotamer libraries 'backbone dependent' and 'backbone independent'. The skeleton-dependent rotamer library allows different rotamers depending on the position of the residue in the skeleton; Thus, for example, some leucine rotamers are allowed if the position is within the α-helix, and other leucine rotamers are allowed if the position is not within the α-helix. The skeleton independent rotamer library uses all the amino acid rotamers in each position. Generally, a backbone independent library is preferred to include the rest of the core because core flexibility is important. However, backbone-independent libraries are more computationally expensive, and therefore a backbone-dependent library is preferred for surfaces and boundary positions. However, this type of library can be used in any position.

[0114] "Variable residue position" as used herein means an amino acid position of a protein to be designed that is not determined in the design method as a specific residue or

-28rotamer, generally a wild type residue or rotamer. It should be noted that even if the position is selected as a variable position, it is possible that some of the methods disclosed herein optimize the sequence so as to select wild-type residues at the variable position. This usually occurs more often for core residues and less regularly for surface residues. In addition, it is also possible to determine amino acid residues as a non-wild type.

[0115] A "fixed residue position" means that the residue has been identified in the three-dimensional structure as a fixed conformation. In some embodiments, the fixed position is left in the original conformation (which may or may not correlate with the particular rotamer of the rotamer library used). Alternatively, residues can be set as non-wild type residues depending on design needs; For example, when known site-directed mutagenesis techniques have shown that a residue is particularly desirable (e.g., to eliminate proteolytic sites or change the specificity of an AARS substrate), the residue can be determined as a specific amino acid. Residues that can be fixed include, but are not limited to, structurally or biologically functional residues, for example, anchor residues.

[0116] In some embodiments, the determined position may be "floated"; the amino acid or analog in this position is determined, but various rotamers of this amino acid or analog are tested. In this embodiment, the variable residues can be at least one or any of 0.1% to 99.9% of the total amount of residues. Thus, for example, it may be possible to change only a few residues (or one), or most residues, with all possibilities in between.

[0117] As used herein, the term "mutant external" refers to a modified molecule (e.g., mutated external tRNA and / or external mutated aminoacyl-tRNA synthetase) that exhibits reduced performance (relative to wild-type or endogenous) for aminoacylation the corresponding wild type amino acid. "Mutant external" refers to the inability or limited performance, e.g. less than 20% yield, less than 10% yield, less than 5% yield, or e.g. less than 1% yield, tRNA and / or RS for operation with the appropriate naturally occurring amino acid in the translation system of interest. For example, an external mutant RS in a translational system of interest aminoacylates any endogenous tRNA of a translation system of interest in a wild type amino acid with reduced or even zero yield relative to the aminoacylation of endogenous tRNA by endogenous RS.

[0118] It should be noted, however, that the outer mutant RS aminoacylates endogenous tRNA with a replacement amino acid (whether natural or non-naturally occurring) with increased efficiency compared to the ability of endogenous RS to aminoacylate endogenous tRNA with a replacement amino acid. Similarly, the mutated external tRNA works towards higher yield towards the replacement amino acid (or this

(An unnatural or other naturally occurring amino acid) than towards the corresponding wild type.

[0119] "Degenerate wobble codon" refers to a codon coding for a natural amino acid, which codon, when present in the mRNA, is recognized by the natural anti-codon tRNA by at least one non-Watson-Crick base pairing or fluctuating (e.g., pairing AC or GU rules). Watson-Crick base pairing refers to base pairing either of GC or AU (RNA or hybrid DNA / RNA) or AT (DNA). When used in the context of base pairing codon mRNA - tRNA anti-codon, Watson-Crick base pairing means that all codon-anti-codon base pairings are mediated by either GC or AU pairing.

[0120] The term "preferentially aminoacylates" refers to yield, e.g., about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 85%, about 90% , about 95%, about 99% or more efficiency. The yield can be measured by how the modified or external mutated aminoacyl-tRNA synthetase aminoacylates the tRNA with a replacement amino acid, whether an unnatural amino acid or other naturally occurring amino acid relative to the corresponding natural amino acid assigned to a particular tRNA, AARS or both. The term "preferentially aminoacylates" can further refer to the performance of the modified or external mutated aminoacyl-tRNA synthetase for aminoacylation or loading of tRNA with any amino acid other than the corresponding natural amino acid assigned to a particular tRNA, AARS, or both. The term "preferentially aminoacylates" may additionally refer to the yield of the modified or external mutated aminoacyl-tRNA synthetase for aminoacylating tRNA with an unnatural amino acid compared to unmodified or naturally occurring AARS.

[0121] It should be noted that the AARS aminoacylation efficiency of tRNA may be correlated with the specificity yield or fidelity of introducing an unnatural amino acid into a target polypeptide or protein. This is due to the operation of protein synthesis machines in that when tRNA is aminoacylated with an amino acid (either wild type or unnatural amino acid), the loaded tRNA is released from the AARS enzyme and the amino acid is introduced into the target polypeptide. When the ability to correct AARS changes, the enzyme will allow the surrogate amino acid to load tRNA and release for introduction into the target protein. Thus, the AARS aminoacylation efficiency directly correlates with the fidelity or specificity of introducing an unnatural amino acid into the target polypeptide.

[0122] The replacement amino acid (whether unnatural or naturally occurring) is then introduced into the growing polypeptide chain with high fidelity, e.g. greater than about 20%, 30%, 40%, 50%, 60%, 75%, 80% , 90%, 95%, or more than about 99% yield for a given codon.

[0123] The term "complementary" refers to elements of the outer mutated pair, the mutated outer tRNA, and the outer mutated synthetase that can

Work together e.g. external mutant synthetase aminoacylates mutated external tRNA.

[0124] The term "derived from" refers to a component that is isolated from the body or isolated and modified, or made, e.g., chemically synthesized using the information of the component from the body.

[0125] The term "translational system" refers to the elements necessary to introduce a naturally occurring or unnatural amino acid into a growing polypeptide chain (protein). For example, the components may include ribosomes, tRNA, synthetase (s), mRNA and the like. The components disclosed herein can be added to the translational system, in vivo or in vitro. The in vivo translation system can be a cell (eukaryotic or prokaryotic). The in vitro translation system can be a cell-free system such as being reconstituted with one of the elements derived from various organisms (purified or recombinantly produced). In some embodiments, the translation system does not include a cell. In some embodiments, the translation system does not include an auxotrophic cell. If the translation system does not include an auxotrophic cell, it may contain another cell or cellular components.

[0126] The term "inactive RS" means a synthetase that has been mutated so that it can no longer aminoacylate its corresponding tRNA with any amino acid, whether natural or not naturally occurring. The term "modified RS" means a synthetase that has been mutated so that it can no longer aminoacylate its corresponding tRNA with the corresponding naturally occurring amino acid, but may be able to aminoacylate its corresponding tRNA with another amino acid, preferably an unnatural amino acid.

[0127] The term "selection factor" refers to an agent that, when present, allows the selection of specific components from a population, e.g., an antibiotic, light wavelength, antibody, nutrient or the like. The selection factor can be changed, e.g. as concentration, intensity, etc.

[0128] The term "positive selection marker" refers to a marker that, if present, e.g., expressed, activated or the like, identifies the organism with a positive selection marker compared to that without a positive selection marker.

[0129] The term "negative selection marker" refers to a marker that, if present, e.g., expressed, activated or similar, allows the identification of an organism that does not possess the desired property (e.g., for organisms that did not possess the desired properties) .

[0130] The term "reporter" refers to an ingredient that can be used to select the ingredients described in the disclosure. For example, the reporter may include green fluorescent protein, firefly luciferase protein, or genes such as β-gal / lacZ (βgalactosidase), Adh (alcohol dehydrogenase) or the like.

[0131] The term "unrecognized efficiently" refers to a yield, e.g., less than about 10%, less than about 5%, or less than about 1%, with which RS from one organism aminoacylates the mutated external tRNA. In some embodiments, the RS may be from the same or a different organism than the mutated external tRNA. In some embodiments, the RS has been modified to aminoacylate the tRNA with a specific amino acid, preferably an unnatural amino acid.

[0132] The term "eukaryote" refers to organisms belonging to the Eucarya phylogenetic domain, such as animals (e.g. mammals, insects, reptiles, birds, etc.), ciliates, plants, fungi (e.g. yeast, etc.), flagellates , microsporidia, protists, etc. In addition, the term "prokaryotes" refers to non-eukaryotic organisms belonging to the phylogenetic domains of proper bacteria (e.g. Escherichia coli, Thermus thermophilus, etc.) and Archaea (e.g. Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium such as Elaloferax volcanii and Halobacterium species NRC-1, A. fulgidus, P. firiosus, P. horikoshii, A. pernix, etc.).

Genetic Code, Host Cells and Degenerate Codons [0133] The standard genetic code of most cells use the following.

Genetic Code Agent

<td>First</td><td>AT</td><td>C</td><td>AND</td><td>G</td><td>final</td>

<td></td><td>phe</td><td>Cheese</td><td>Tyr</td><td>Cys</td><td>AT</td>

<td></td><td>phe</td><td>Cheese</td><td>Tyr</td><td>Cys</td><td>c</td>

<td>AT</td><td>Leu</td><td>Cheese</td><td>Stop</td><td></td><td></td>

<td>(Ocher)</td><td>Stop</td><td></td><td></td><td></td><td></td>

<td>(Umber)</td><td>AND</td><td></td><td></td><td></td><td></td>

<td></td><td>Leu</td><td>Cheese</td><td>Stop</td><td></td><td></td>

<td>(Amber)</td><td>Trp</td><td>G</td><td></td><td></td><td></td>

<td></td><td>Leu</td><td>Pro</td><td>His</td><td>Arg</td><td>at</td>

<td></td><td>Leu</td><td>Pro</td><td>His</td><td>Arg</td><td>c</td>

<td>C</td><td>Leu</td><td>Pro</td><td>Gin</td><td>Arg</td><td>AND</td>

<td></td><td>Leu</td><td>Pro</td><td>Gin</td><td>Arg</td><td>G</td>

<td></td><td>How much</td><td>Thr</td><td>own</td><td>Cheese</td><td>AT</td>

<td>AND</td><td>lie</td><td>Thr</td><td>own</td><td>Cheese</td><td>C</td>

<td></td><td>How much</td><td>Thr</td><td>lys</td><td>Arg</td><td>AND</td>

<td></td><td>Underworld</td><td>Thr</td><td>lys</td><td>Arg</td><td>G</td>

<td></td><td>val</td><td>ala</td><td>Asp</td><td>Gly</td><td>AT</td>

<td>First</td><td>AT</td><td>C</td><td>AND</td><td>G</td><td>final</td>

<td>G</td><td>val</td><td>ala</td><td>Asp</td><td>Gly</td><td>C</td>

<td></td><td>val</td><td>ala</td><td>Glu</td><td>Gly</td><td>AND</td>

<td></td><td>Yal</td><td>ala</td><td>Glu</td><td>Gly</td><td>G</td>

[0134] The genetic code is degenerated in that the protein biosynthesis machine uses 61 mRNA sense codons to drive polymerization based on a matrix of 20 amino acid monomers. (Crick et al., Nature 192: 1227, 1961). Only two amino acids, i.e. methionine and tryptophan, are encoded by unique mRNA triplets.

[0135] The standard genetic code applies to most, but not all cases. Exceptions were found in the mitochondrial DNA of many organisms and in the nuclear DNA of several lower organisms. Some examples are given in the table below.

Examples of non-standard genetic codes ._

<td>mitochondria</td><td>vertebrata</td><td>UGC—&gt; Trp; AGA, AGG STOP</td>

<td></td><td>invertebrates</td><td>UGA—&gt; Trp; AGA, AGG Ser</td>

<td></td><td>Yeast</td><td>UGC—&gt; Trp; CUN Thr</td>

<td></td><td>protists</td><td>UGA — ► Trp;</td>

<td>Kernel</td><td>Bacteria</td><td>GUG, UUG, AUU, CUG initiation</td>

<td></td><td>Yeast</td><td>CUG - + Cheese</td>

<td></td><td>ciliates</td><td>UAA, UAG Gin</td>

<td colspan="3">* Plant cells use the standard genetic code in both mitochondria and the nucleus.</td>

[0136] NCBI (National Center for Biotechnology Information) maintains a detailed list of standard genetic code and genetic codes used in various organisms, including vertebrate mitochondrial code; yeast mitochondrial code; mitochondrial code for molds, protozoa and jamophylls and the Mycoplasma / Spiroplasma code; invertebrate mitochondrial code; nuclear code of ciliates, dasycladaceans and hexamites; Mitochondrial code for echinoderms and flatworms; euplotide nuclear code; bacterial and plant bottom code; alternative yeast nuclear code; mitochondrial code of żachw; alternative genetic code for flatworms; Blepharisma nuclear code; genetic code of true green algae; mitochondrial code Scenedesmus obliąuus mitochondrial code; Thraustochytrium mitochondrial code (all incorporated by reference). They are based primarily on a review by Osawa et al., Microbiol. Rev. 56: 229264, 1992, and Jukes and Osawa, Comp. Biochem. Physiol. 106B: 489-494, 1993.

Host Cells

[0137] Some of the methods disclosed herein can be used in a cell, which allows levels of protein production to be made for practical purposes. In preferred embodiments, the cells are used for cultured cells (i.e., the cells may be grown under laboratory conditions). Suitable cells include mammalian (human or non-human mammal) cells, bacterial cells, and insect cells, etc.

[0138] One example includes PFENEX ™ technology, which is a cell line using a Pseudomonas fluorescens-based cell line that increases cell expression, while maintaining some solubility and activity characteristics due to the use of different pathways in the metabolism of some sugars relative to E.coli.

[0139] In addition, other auxotrophic host cell lines include the KIO-based auxotrophic Phy strain (AF), the dual auxotrophic Phe / Trp (AFW) strains, the triple auxotrophic Phe / Trp / Lys (AFWK) strains, the Met auxotroph (M15MA on background Ml5) ), and the strain DH10B based AF.

[0140] Cells that can be used in carrying out some of the embodiments disclosed herein include auxotrophic host cells (prokaryotic or eukaryotic). Auxotrophic cells can have single, double, triple, quadruple or more levels of auxotrophy (each level of auxotrophy defines an organic compound that the body cannot synthesize and must be delivered to the cell). Certain embodiments disclosed herein do not explicitly use an auxotrophic host cell. Unless an auxotrophic host cell is used, another cell or cellular components may still be used to implement some of the embodiments disclosed herein. Other embodiments may use one, two, three or more different auxotrophic host cells, which may be from the same or different strains or organisms.

[0141] Host cells are modified by genetic engineering (e.g., transformed, transduced or transfected) vectors of the present invention, which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, bacterium, virus, naked polynucleotide or conjugated polynucleotide. These vectors are introduced into cells and / or microorganisms by standard methods that include electroporation (From et al., PNAS. USA 82, 5824 (1985)), infection by viral vectors, ballistic penetration at high velocity of small nucleic acid particles, or in matrix of small beads or particles, either on the surface (Klein et al., Nature 327, 70-73 (1987)). Berger, Sambrook, and Ausubel provide many suitable transformation methods.

[0142] These engineered modified host cells may be cultured in conventional modified media for activities such as, for example, screening steps, promoter activation or selection of transformants, respectively. These cells can optionally be cultured in transgenic organisms.

[0143] Certain embodiments disclosed herein further include methods for screening modified AARS and / or modified tRNAs. For example, in one embodiment, the yeast PheRS library is screened through a double sieve to detect a high level of unnatural amino acid introduction or the incorrect introduction of non-Phe natural amino acids leads to major conformational changes or GFP development. The yeast PheRS library cells are thus subjected to high throughput screening based on flow cytometry analysis (FACS). First, the cells of the yeast PheRS library are expressed in the presence of 2Nal and low fluorescent cells indicating higher incorporation of 2Nal or other natural amino acids are harvested by FACS. The yeast PheRS library cells are then expressed without 2Nal. Bright cells are harvested to remove yeast PheRS variants that may mistakenly introduce other natural amino acids. In an exemplary embodiment, two screening cycles gave mutated yeast PheRS with mutations in N412G, S418C, T415G and S437F, which had low fluorescence in the presence of 2Nal and high fluorescence in the absence of 2Nal. This technique allows the introduction of 2Nal at the UUU codon, increasing to about 90%.

[0144] Other useful references, for example, for the isolation and culture of cells (e.g., for the subsequent isolation of nucleic acid) include Freshney (1994) Culture of Animal Cells, and the Manual of Basic Technique, third edition, Wiley-Liss, New York and literature cited there; Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley &amp; Sons, Inc. New York, NY; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York) and Atlas i Parks (eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.

[0145] Several known methods for introducing nucleic acids into target bacterial cells are available, each of which may be used in some of the embodiments disclosed herein. These include: fusion of recipient cells with bacterial protoplasts containing DNA, electroporation, bullet bombardment and infection with viral vectors, etc. Bacterial cells can be used to amplify many plasmids containing DNA constructs of some of the embodiments disclosed herein. Bacteria are grown to log phase and plasmids within bacteria can be isolated by a variety of different methods known in the art (see, for example, Sambrook). In addition, many kits for the purification of plasmids from bacteria are commercially available (see, e.g., EASYPREP ™, FLEXIPREP ™, both from Pharmacia Biotech; STRATACLEAN ™, from Stratagene; and, QIAPREP ™ from Qiagen). Isolated and purified plasmids are further processed to produce other plasmids used for transfection of cells or introduced into related vectors to infect organisms.

[0146] Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulating the expression of a particular target nucleic acid. The vectors optionally contain generic expression cassettes containing at least one independent terminator sequence, enabling sequences

Replication in eukaryotic cells of the cassette in eukaryotes or prokaryotes or both (e.g. shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems. Vectors are suitable for replication and integration in prokaryotes, eukaryotes, and preferably both. See Giliman &amp; Smith, Gene 8:81 (1979); Roberts, et al., Nature, 328: 731 (1987); Schneider, B., et al., Protein Expr. Purif. 6435: 10 (1995); Ausubel, Sambrook, Berger (all supra). A catalog of Bacteria and Bacteriophages useful for cloning is provided, e.g., by ATCC, e.g., The ATCC Catalog of Bacteria and Bacteriophage (1992) Ghema et al. (Eds.) Published by ATCC. Additional basic sequencing, cloning, and other aspects of molecular biology and basic theoretical considerations can also be found in Watson et al. (1992) Recombinant DNA 2nd Edition Scientific American Books, NY.

Degenerate Codon Selection [0147] As described above, all amino acids except for methionine and tryptophan are encoded by more than one codon. According to some methods disclosed herein, the codon in the genome that is typically used to encode a natural amino acid is reprogrammed, in part, by transcription or translation machinery to encode the amino acid analog instead. The amino acid analog may be a naturally occurring or canonical amino acid analog. In a preferred embodiment, the amino acid analog is not a canonically coded amino acid.

[0148] The thermodynamic stability of a codon anti-codon pair can be predicted or determined experimentally. According to some embodiments, it is preferred that the mutated external tRNA interacts with a degenerate codon with affinity (at 37 ° C) at least about 1.0 kcal / mol more strongly, even more preferably 1.5 kcal / mol more strongly, and even more preferably over 2.0 kcal / mole more strongly than the natural tRNA in the cell would recognize the same sequence. These values ​​are known to those skilled in the art and can be determined by thermal denaturation experiments (see, e.g., Meroueh and Chow, Nucleic Acids Res. 27: 1118, 1999).

[0149] The following table contains some of the known anti-codon sequences for E. coli. In general, for any organism, the tRNA anti-codon sequence can be routinely determined using technologies known in the art. For example, any tRNA gene can be amplified, for example, PCR. Sequencing can be performed to determine the exact sequences of the anti-codon loop. Alternatively, a biochemical binding assay can be used to determine the binding affinity of purified tRNA for one of 2-6 possible codons. The codon that binds tRNA with the highest specificity / affinity presumably shows a clean WatsonCrick alignment at all three codon positions, thus determining the sequence of the anti-codon loop.

[0150] Generally, the wobble principle in the anti-codon loop tends to be G or U (and not A or C).

Degenerate codons for E. coli

<td>Amino acid ace</td><td>Antykodo n</td><td>evaporation e principles at the 3rd Principle</td><td>Kodo n</td><td>Amino acid ace</td><td>Antykodo n</td><td>evaporation e rules</td><td>Kodo n</td>

<td rowspan="4">ala</td><td rowspan="2">GGC</td><td>TOILETS¹</td><td>GCC</td><td rowspan="2">His</td><td rowspan="2">GUG</td><td>TOILETS</td><td>CAC</td>

<td>Wobble2</td><td>GCU</td><td>wobble</td><td>CAU</td>

<td rowspan="2">UGC</td><td>TOILETS</td><td>GCA</td><td rowspan="2">How much</td><td rowspan="2">GAU</td><td>TOILETS</td><td>AUC</td>

<td>wobble</td><td>GCG</td><td>wobble</td><td>AUU, AUA</td>

<td rowspan="2">Asp</td><td rowspan="2">GUC</td><td>TOILETS</td><td>GAC</td><td rowspan="2">Leu</td><td rowspan="2">GAG</td><td>TOILETS</td><td>cuc, CUA, MOVE, UUC, UUG</td>

<td>wobble</td><td>GAU</td><td>wobble</td><td>CUU</td>

<td rowspan="2">own</td><td rowspan="2">GUU</td><td>TOILETS</td><td>AAC</td><td rowspan="2">lys</td><td rowspan="2">uuu</td><td>TOILETS</td><td>AAA</td>

<td>wobble</td><td>AAU</td><td>wobble</td><td>AAG</td>

<td rowspan="2">Cys</td><td rowspan="2">GCA</td><td>TOILETS</td><td>UGC</td><td rowspan="2">phe</td><td rowspan="2">GAA</td><td>TOILETS</td><td>UUC</td>

<td>wobble</td><td>UGU</td><td>wobble</td><td>uuu</td>

<td rowspan="2">Glu</td><td rowspan="2">UUC</td><td>TOILETS</td><td>GAA</td><td rowspan="2">Cheese</td><td rowspan="2">GGA</td><td>TOILETS</td><td>UUC, AGU</td>

<td>wobble</td><td>GAG</td><td>wobble</td><td>UCU, AGC UCA UCG</td>

<td rowspan="2">Gly</td><td rowspan="2">GCC</td><td>TOILETS</td><td>GGC, GGA GGG</td><td rowspan="2">Tyr</td><td rowspan="2">GUA</td><td>TOILETS</td><td>UAC</td>

<td>wobble</td><td>GGU</td><td>wobble</td><td>UAU</td>

<td>Underworld</td><td></td><td>TOILETS</td><td>AUG</td><td rowspan="2">Thr</td><td rowspan="2"></td><td>TOILETS</td><td>ACC, ACA ACG</td>

<td>Gin</td><td></td><td>TOILETS</td><td>CAA CAG</td><td>wobble</td><td>ACU</td>

<td>Arg</td><td></td><td>TOILETS</td><td>AGA,</td><td>Pro</td><td></td><td>TOILETS</td><td>CCC</td>

<td rowspan="3"></td><td rowspan="3"></td><td></td><td>AGG CGU CGG</td><td></td><td></td><td></td><td>CCA CCG</td>

<td rowspan="2">wobble</td><td rowspan="2">CGC, CGA</td><td rowspan="2">Trp</td><td rowspan="2"></td><td>wobble</td><td>CCU</td>

<td>TOILETS</td><td>UGG</td>

<td rowspan="2">STOP</td><td rowspan="2"></td><td>TOILETS</td><td>UGA UAA</td><td rowspan="2">val</td><td rowspan="2"></td><td>TOILETS</td><td>GUC, GUA</td>

<td>wobble</td><td>UAG</td><td>wobble</td><td>GUU, GUG</td>

<td colspan="8">¹ Pairing Watson-Crick rules 2 Hesitating pairing of rules</td>

[0151] When a single tRNA recognizes a codon due to the excellent complementary interaction between the tRNA anti-codon and one codon, this is called Watson-Crick base pairing. When a single tRNA recognizes a second degenerate codon, this is called wobble or custom base pairing. In certain embodiments disclosed herein, a new tRNA may be constructed with an anti-codon sequence that is ideally complementary to the degenerate codon or codons for unnatural amino acids, thereby utilizing the base or Watson-Crick pairing. Similarly, a new AARS can be constructed that uses a replacement amino acid (other than wild type, can be another naturally occurring amino acid or unnatural amino acid) to aminoacylate the corresponding tRNA. This can be done in addition to or instead of modifying the tRNA molecule to introduce a replacement amino acid.

[0152] The modified AARS can be changed in such a way that the binding efficiency of the unnatural amino acid or other selected naturally occurring amino acid is greater than the binding capacity of the modified AARS to the corresponding naturally occurring amino acid. Thus modified AARS can advantageously bind a non-natural amino acid to attach to the tRNA even in the presence of a naturally occurring amino acid that corresponds to AARS in its unchanged state. This "reprogramming" of the aminoacyl-tRNA synthetase allows the introduction of an unnatural amino acid into the polypeptide at lower levels of incorrect introduction of other amino acids to the desired site.

[0153] "Reprogramming" further, may allow the use of external or modified mutant synthetase with high levels of introduction into standard host cells, without the need for auxotrophic host cells and without or depletion of media from the corresponding naturally occurring amino acid. Therefore, while some embodiments disclosed herein can be carried out using an auxotrophic host cell, some other embodiments can be carried out without using an auxotrophic host cell. When not using auxotrophic

- host cells In order to perform certain embodiments, another host cell may be used, cell components may be used, or a completely cell free system may be used.

[0154] When a cell has many tRNA molecules for a given amino acid and one tRNA has an anti-codon sequence that is perfectly complementary to the degenerate codon selected, the gene encoding tRNA can be turned off by any means available to the skilled person, for example, site-directed mutagenesis or the removal of such a gene or gene promoter sequence. Gene expression can also be turned off by any RNA antisense or interference technique.

Unnatural or Non-Natural Amino Acids [0155] The first step in protein engineering is usually to choose a set of unnatural or non-naturally occurring amino acids that have the desired chemical properties. The choice of unnatural amino acids depends on the predetermined chemical properties that you would like to have and the modifications that you would like to perform on the target protein. Unnatural amino acids after selection can either be purchased from sellers or chemically synthesized.

[0156] A wide variety of unnatural or non-naturally occurring amino acids can be used in the methods disclosed herein. An unnatural amino acid can be selected based on the desired properties of the unnatural amino acid, e.g. the action of an unnatural amino acid, such as modifying the biological properties of a protein such as toxicity, biodistribution, immunogenicity or half-life, structural properties, spectroscopic properties, chemical and / or photochemical properties, catalytic properties, ability to interact with other molecules (covalently or non-covalently) or similar.

[0157] As used herein, "unnatural amino acid" refers to any amino acid, modified amino acid or amino acid analogue other than selenocysteine ​​and twenty genetically coded alpha amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine , histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine. The general structure of alpha-amino acids is represented by Formula I:

<img file="PL1999259T3_D0001.tif" />

[0158] The unnatural amino acid usually has any structure of general formula I, wherein the R group is any substituent other than that used in the twenty natural amino acids. See, e.g., any biochemical text, such as Biochemistry by L. Stryer,

3. Edition, 1988, Freeman and Company, New York, for twenty natural structures

-39aminokwasów. Note that the unnatural amino acids disclosed herein may be naturally occurring compounds other than the twenty alpha-amino acids above. Because the unnatural amino acids disclosed herein usually differ from the natural amino acids only in the side chain, the unnatural amino acids form bonds with other amino acids, e.g., natural or unnatural, in the same way they are formed in naturally occurring proteins. However, unnatural amino acids have side chain groups that distinguish them from natural amino acids. For example, R in Formula I optionally includes alkyl-, aryl-, aryl halide, vinyl halide, alkyl halide, acetyl, ketone, aziridine, nitrile, nitro, halide, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano -, halo-, hydrazide, alkenyl, alkynyl, ether, thioether, epoxy, sulfone, boric acid, boronic acid ester, borate, phenylboronic acid, thiol, selenium, sulfonyl, borate, boronate, phospho, phosphono, phosphine, heterocyclic, pyridyl, naphthyl, benzophenone, such a forced ring, like cyclooctyne; thioester, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amine, carboxylic acid, alpha-keto carboxylic acid, alpha or beta-unsaturated acids and their amides, glyoxylamide or organosilane group or the like, or any combination thereof.

[0159] Aryl substitutions can occur in various positions, for example ortho, meta, para, and with one or more functional groups located on the aryl ring. Other unnatural amino acids of interest include, but are not limited to, amino acids containing a photoactivated crosslinker, spin-labeled amino acids, dye-labeled amino acids, fluorescent amino acids, metal binding amino acids, metal containing amino acids, radioactive amino acids, new functional amino acids, altered amino acids hydrophilicity, hydrophobicity, polarization, or hydrogen binding capacity, amino acids, which covalently or non-covalently interact with other molecules, photoclatted amino acids ( photocaged) and / or photoisomerized amino acids containing biotin or a biotin analog, glycosylated amino acids such as serine-substituted sugar, other carbohydrate-modified amino acids, keto-containing amino acids, polyethylene glycol or polyether-containing amino acids, polyalcohol or polysaccharide, and amino acids that may undergo metatates , amino acids that may undergo cycloaddition, heavy-substituted amino acids, chemically cleavable and / or photo-cleavable amino acids, amino acids with elongated side chains compared to natural amino acids, e.g. polyethers or long chain hydrocarbons, e.g. greater than about 5 or more than about 10 carbons, carbon-linked sugar-containing amino acids, active redox amino acids, amino acid containing amino acid, amino acid containing amino acid and amino acids containing one or more toxic moieties.

[0160] In addition to unnatural amino acids that contain new side chains, unnatural amino acids optionally also contain modified backbone structures, e.g., as shown in structures of Formula II and III:

40R

<img file="PL1999259T3_D0002.tif" />

Wzoril Formula Ul wherein Z typically contains OH, NH₂, SH, NH₂O-, NH-R ', R'NH-, R'S- or S-R'-; X and Y, which may be the same or different, usually contain S, N or O, and R and R ', which are optionally the same or different, are usually selected from the same list of ingredients for the group R described above for unnatural amino acids Formula I and hydrogen or (CH₂)_x or natural amino acid side chains. For example, the unnatural amino acids disclosed herein optionally have amino or carboxy substitutions as shown in Formulas II and III. Unnatural amino acids of this type include, but are not limited to, α-hydroxy acids, α-thio acids, amino-thiocarboxylates, or α-α-disubstituted amino acids, with side chains corresponding to, e.g., twenty natural amino acids or unnatural side chains. They may include, but are not limited to, β-amino acids or γ-amino acids such as substituted β-alanine and γ-aminobutyric acid. In addition, substitution or modification at α-carbon optionally includes L or D isomers such as Dglutamate, D-alanine, D-methyl-O-tyrosine, aminobutyric acid and the like. Other structural alternatives include cyclic amino acids such as proline analogues and proline analogues with a 3, 4, 6, 7, 8 and 9-membered ring. Some unnatural amino acids such as aryl halides (p-bromo-phenylalanine, p-iodophenylalanine) provide universal palladium-catalyzed cross-coupling reactions with ethylene or acetylenes that allow the formation of carbon-carbon, carbon-nitrogen and carbon-oxygen bonds between halides aryl and various coupling partners.

[0161] For example, many unnatural amino acids are based on natural amino acids such as tyrosine, glutamine, phenylalanine, and the like. Tyrosine analogs include para-substituted tyrosines, ortho-substituted tyrosines and meta-substituted tyrosines, wherein the substituted tyrosine contains an acetyl group, a benzoyl group, an amino group, a hydrazine, a hydroxyamino group, a thiol group, a carboxyl group, an isopropyl group, a methyl group, C6-C20 straight or branched chain hydrocarbon, saturated or unsaturated hydrocarbon, O-methyl group, polyether group, nitro group or the like. In addition, multi-substituted aryl rings are also contemplated. Glutamine analogs include, but are not limited to, α-hydroxy derivatives, β-substituted derivatives, cyclic derivatives, and amide-substituted glutamine derivatives. Exemplary phenylalanine analogs include, but are not limited to, meta-substituted phenylalanines, wherein the substituent includes a hydroxyl group, methoxy group, methyl group, allyl group, acetyl group or the like.

[0162] Specific examples of unnatural amino acids include, but are not limited to, o, mi / or p forms of amino acids or amino acid analogs (unnatural amino acids), including homoallylglycine, cis- or trans-crotylglycine, 6,6,6-acid trifluoro-2-aminohexanoic acid, 2-aminopheptanoic acid, norvaline, norleucine, O-methyl-L-tyrosine, o-, m- or p-methyl-phenylalanine, Ο-4-allyl-L-tyrosine, 4-propyl-Ltyrosine, tri -O-acetyl-GlcNAcP-serine, L-Dopa, fluorinated phenylalanine, isopropyl-phenylalanine, p-azidophenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonothyrosine, p-iodo-phenylalanine, o-, m- or pbromophenylalanine 4-pyridylalanine, p-idiophenylalanine, diaminobutyric acid, aminobutyric acid, benzofuranylalanine, 3-bromo-tyrosine, 3- (6-chloroindolyl) alanine, 3- (6-bromoindolyl) alanine, 3- (5-bromonindolyl) alanine, pchlorine -ethynyl-phenylalanine, p-propargyl-oxy-phenylalanine, m-ethynylphenylalanine, 6-ethynyl-tryptophan, 5-ethynyl-tryptophan, (R) -2-amino-3- (4-ethynyl-1H-pyrol-3-yl) propanoic acid, azidororleucine, azidohomoalanine, p-acetylphenylalanine, pamino L-phenylalanine, homopropargylglycine, p-ethyl-phenylalanine, p-ethynylphenylalanine, p-propargyl-oxyphenylalanine, isopropyl-L-phenylalanine, 3- (2-naphthyl) alanine, 3- (1-naphthyl) alanine, 3-idio -tyrosine, O-propargyl-tyrosine, homoglutamine, Ο-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, 3-nitro-L-tyrosine, tri-Oacetyl-GlcNAcP-serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, peptide-L-phenylalanine, p-acyl-L-phenylalanine, p-acetyl-L-phenylalanine, m-acetyl-L-phenylalanine, selenomethionine , telluromethionine, selenocysteine, alkynyl phenylalanine, Oallyl-L-tyrosine, O- (2-propynyl) -L-tyrosine, p-ethylthiocarbonyl-L-phenylalanine, p- (3-oxobutanoyl) -L-phenylalanine, p-benzoyl-L-phenylalanine phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, homopropargylglycine, azidohomoalanine, p-iodo-phenylalanine, p-bromoL-phenylalanine, dihydroxy-phenylalanine, dihydroxyl-L-phenylalanine, p-nitro-L-phenylalanine, m-methoxy-L-phenylalanine, p-iodo-phenylalanine, p-bromophenyl L-phenylalanine and isopropyl-L-phenylalanine, trifluoroleucine, norleucine, 4-, 5- or 6-fluoro-tryptophan, 4-aminotriptophan, 5-hydroxytryptophan, biocyte, aminoxyoxyacetic acid, m-hydroxyphenylalanine, m-allylphenylethalate β-GlcNAc-serine, α-GalNAc-threonine, p-acetoacetylphenylalanine, para-halo-phenylalanine, selenomethionine, ethionine, S-nitroso-homocysteine, thia-proline, 3-thienyl-alanine, homo-allyl-glycine, trifluoroisoleucine, and transfluoroisoleucin -amino-4-hexene, 2-butynyl-glycine, allyl-glycine, para-azido-phenylalanine, para-cyanophenylalanine, para-ethynyl-phenylalanine, hexafluoroleucine, 1,2,4-triazole-3-alanine, 2-fluoro-histidine -methylhistidine, 3-methyl-L-histidine, β-2-thienyl-L-alanine, β- (2-thiazolyl) -DL-alanine, homopropargylglycine (HPG) and azidohomoalanine (AHA) and the like. The structures of various non-limiting unnatural amino acids are shown in the figures, e.g., FIG. 29, 30 and 31 in US 2003/0108885 A1, all of which is incorporated herein by reference.

[0163] Tyrosine analogs include para-substituted tyrosines, ortho-substituted tyrosines, and meta-substituted tyrosines, wherein the substituted tyrosine contains an acetyl group, a benzoyl group, an amino group, hydrazine, hydroxyamine, a thiol group, a carboxyl group,

Isopropyl group, methyl group, C6-C20 straight chain or branched hydrocarbon, saturated or unsaturated hydrocarbon, O-methyl group, polyether group, nitro group, or the like. In addition, multi-substituted aryl rings are also contemplated. The glutamine analogs of the invention include, but are not limited to, α-hydroxy derivatives, β-substituted derivatives, cyclic derivatives and amide-substituted glutamine derivatives. Exemplary phenylalanine analogs include, but are not limited to, meta-substituted phenylalanines, wherein the substituent includes a hydroxy group, methoxy group, methyl group, allyl group, acetyl group or the like.

[0164] In addition, other examples optionally include (but are not limited to) an unnatural amino acid tyrosine analogue; unnatural glutamine amino acid analogue; an unnatural amino acid analog of phenylalanine; an unnatural amino acid analogue of serine; an unnatural amino acid threonine analogue; alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronic, phospho, phosphono, phosphine, heterocycle, enone, imine, aldehyde, hydroxylamine, keto, or amine substituted amino acid, or any combination thereof; amino acid with photoactivated crosslinker; spin-labeled amino acid; fluorescent amino acid; amino acid with a new functional group; amino acids that covalently or non-covalently interacts with another molecule; metal binding amino acid; metal containing amino acid; radioactive amino acid; photoclassified amino acid; photoisomerizable amino acid; biotin or an amino acid containing a biotin analogue; a glycosylated or carbohydrate modified amino acid; keto containing amino acid; amino acid containing polyethylene glycol; polyether containing amino acid; heavy-substituted amino acid; chemically cleavable or photo-cleavable amino acid; elongated side chain amino acid; an amino acid containing a toxic group; sugar substituted amino acid, e.g. sugar substituted serine or the like; an amino acid containing a carbon bonded sugar; active redox amino acid; a-hydroxy acid; amino acid containing amino acid; a, a disubstituted amino acid; β-amino acid; and cyclic amino acid.

[0165] Typically, the unnatural amino acids used herein for certain embodiments can be selected or designed to provide additional properties not available in twenty natural amino acids. For example, unnatural amino acids are optionally intended or selected to modify the biological properties of the protein, e.g., into which they are introduced. For example, the following properties are optionally modified by incorporating an unnatural amino acid into a protein: toxicity, biodistribution, solubility, stability, e.g. thermal, hydrolytic, oxidative, resistance to enzymatic degradation and the like, ease of purification and processing, structural properties, spectroscopic properties, chemical and / or photochemical properties, catalytic activity , redox potential, half-life, ability to react with other molecules, e.g. covalently or non-covalently, and the like.

[0166] Other examples of amino acid analogs optionally include (but are not limited to) an unnatural amino acid tyrosine analog; unnatural analogue of the amino acid glutamine; an unnatural amino acid analog of phenylalanine; an unnatural amino acid analogue of serine; an unnatural amino acid analogue of threonine; alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronic, phospho, phosphono, phosphine, heterocycle, enone, imine, aldehyde, hydroxylamine, keto, or amine substituted amino acid, or any combination thereof; amino acid with photoactivated crosslinker; spin-labeled amino acid; fluorescent amino acid; amino acid with a new functional group; amino acids that covalently or non-covalently interacts with another molecule; metal binding amino acid; metal containing amino acid; radioactive amino acid; photoclassified amino acid; photoisomerizable amino acid; biotin-containing amino acid or biotin analogue; a glycosylated or carbohydrate modified amino acid; keto containing amino acid; amino acid containing polyethylene glycol; polyether containing amino acid; heavy-substituted amino acid; chemically cleavable or photo-cleavable amino acid; elongated side chain amino acid; an amino acid containing a toxic group; sugar substituted amino acid, e.g. sugar substituted serine or the like; an amino acid containing a carbon bonded sugar; active redox amino acid; ahydroxy acid; amino acid containing thioamino acid; a, a disubstituted amino acid; βaminokwas; and a cyclic amino acid other than proline.

Aminoacyl-tRNA Synthetase [0167] Aminoacyl-tRNA Synthetase (used interchangeably herein with AARS, RS or "synthetase") used in some of the methods disclosed herein may be a naturally occurring synthetase derived from an organism, either the same (homologous) or different (heterogeneous ), a mutated or modified synthetase or designed synthetase.

[0168] The synthetase used may recognize the desired (unnatural) amino acid analogue selectively over related amino acids. For example, when the amino acid analog to be used is structurally related to a naturally occurring amino acid, the synthetase should attach to the mutated outer tRNA molecule the desired amino acid analog in a yield at least substantially equivalent to, and more preferably at least twice, 3 times, 4 times, 5 times, or greater than that of the naturally occurring amino acid. However, in cases where a well-defined protein product is not required, the synthetase may attenuate the specificity of amino acid loading. In such an embodiment, a mixture of mutated external tRNAs with different amino acids or analogs can be produced.

[0169] In certain embodiments, it is preferred that the synthetase exhibits activity for both the amino acid analog and the amino acid that is encoded by the corresponding codon of the tRNA molecule.

[0170] Synthetase can be obtained using a variety of techniques known to those of skill in the art, including combinations of such techniques as, for example, calculation methods, selection methods, and the introduction of synthetases from other organisms (see below).

[0171] In certain embodiments, synthetases that efficiently take up tRNA molecules that are not loaded by host cell synthetases can be used or developed. For example, suitable pairs can generally be developed as part of synthetase modifications from various organisms different from the host cell. In certain embodiments, a synthetase can be developed by selection procedures. In certain embodiments, a synthetase can be designed using computational methods such as those described in Datta et al., J. Am. Chem. Soc. 124: 5652-5653, 2002 and in US Patent No. 7,139,665, hereby incorporated by reference. Computational Design of AARS [0172] Specifically, in one embodiment, the subject method depends in part on the design and engineering of natural AARS into a modified form that has free substrate specificity such that it can capture non-canonical amino acid analogs as a substrate and load modified tRNA (with its altered anti-codon) with such a non-canonical amino acid. The following chapters briefly describe a method for producing such a modified AARS, which method is described in more detail in US Patent No. 7,139,665, the entire contents of which are incorporated herein by reference.

[0173] Briefly, the methods of some of the embodiments described herein relate to computational tools to modify the substrate specificity of AminoAcyl-tRNA Synthetases (AARS) as a result of mutations to allow the enzyme to more efficiently use the amino acid analog (s) in protein translation systems, in in vitro, in whole cells or in other translation systems. A feature of some embodiments includes the systematic redesign of the AARS enzyme substrate binding site to facilitate the use of unnatural substrates in a peptide or protein translation reaction that the enzyme catalyzes.

[0174] According to one method, the artificial amino acid rotamer library is created by changing its torsion angle to form rotamers that match the binding pocket of the natural substrate. The geometrical orientation of the amino acid analog skeleton is determined by the crystallographic orientation of the skeleton of the natural substrate in the crystal structure. An organism-specific crystallographic structure of an amino acid synthetase may be used, or a homologous structure from another organism may be used depending on structural similarity. Amino acids in the synthetase binding pocket that interact with the analogue side chain may differ in identity and conformation of rotamers in subsequent protein design calculations.

[0175] One such protocol also uses computational methods to increase the interaction between substrate and protein positions. This is done by scaling the energy in pairs between the substrate and the amino acids allowed for the designed

-45 positions on protein in energy calculations. In optimization calculations, where protein-substrate interactions are scaled up relative to interactions within the protein, the sequence selection is pressed toward the selection of those amino acids that have a favorable interaction with the substrate.

[0176] The described method helped build a new modified form of E. coli phenylalanyl-tRNA synthetase, based on the known structure of the related PheRS (/ PheRS) Thermus thermophilus. The new modified form of E. coli phenylalanyl-tRNA (ePheRS) synthetase allows for efficient in vivo incorporation of reactive aryl ketone functionality into recombinant proteins. In addition, the modified tryptophanyl-tRNA synthetase was modified in a similar manner and showed the ability to accept unnatural amino acid analogs in polypeptides in place of the naturally occurring tryptophan. The results described here also indicate the overall power of protein computational design in the development of aminoacyl-tRNA synthetase to activate and charge unnatural amino acids.

A. Available Sequence and Structural Information for tRNA Synthetases [0177] Translation of the protein on the mRNA matrix is ​​carried out by ribosomes. During the translation process; each tRNA is matched with its amino acid long before it reaches the ribosome. The alignment is done by a set of enzymes known as aminoacyl-tRNA synthetases (AARS). These enzymes attach the appropriate amino acid to each tRNA, which will allow each tRNA to be correctly translated from the DNA genetic code (and mRNA transcribed from DNA) into the amino acid code of the protein.

[0178] Most cells produce twenty different aminoacyl-tRNA synthetases, one for each type of amino acid. Each of these twenty enzymes is optimized for function by its own amino acid and a set of tRNA molecules suitable for that amino acid. Aminoacyl-tRNA synthetases must perform their tasks with high accuracy. Many of these enzymes recognize their tRNA molecules with anti-codons. These enzymes make one mistake out of 10,000. For most amino acids, this level of accuracy is not too difficult to achieve because most amino acids are completely different from each other.

[0179] In the present method, an accurate description of the AARS binding pocket for tRNA is important for the computational design approach because it depends on the crystal structure of the protein skeleton descriptions, although in many cases it is not entirely acceptable to use the crystal structure of a homologous protein (on example, homologue from related species) or even a conserved domain to provide a description of the crystallographic structure of the binding pocket. The crystal structure also determines the orientation of the natural amino acid substrate in the synthetase binding pocket as well as the relative position of the amino acid substrate to the synthetase residues, especially those residues in and around the binding pocket. To design the binding pocket for these analogs, it is preferred that these analogs bind to the synthetase in the same direction as the natural amino acid substrate, since this orientation may be important for the adenylation step.

[0180] AARS can be derived from any organism, including prokaryotes and eukaryotes, with enzymes from bacteria, fungi, extremophiles such as archebacteria, worms, insects, fish, amphibians, birds, animals (in particular mammals, especially humans) ) and all possible plants.

[0181] As described above, most cells produce twenty different aminoacyl-tRNA synthetases, one for each type of amino acid. Some preferred synthetases are known, including: yeast phenylalanyl-tRNA synthetase (Kwon et al., J. Am. Chem. Soc. 125: 7512-7513, 2003); Methonococcus jannaschii tyrosyl tRNA synthetase (Wang et al., Science 292, 498-500, 2001); and yeast tyrosyl tRNA synthetase (Ohno et al., J. Biochem. 130, 417-423, 2001). In fact, crystal structures of almost all 20 different AARS enzymes are currently available at Brookhaven Protein Data Bank (PDB, see Bernstein et al., J. Mol. Biol. 112: 535-542, 1977). A list of all AARS with solved crystal structures up to April 2001 is available on the PDB website. For example, the crystal structure of Thermus Aquaticus Phenylalanyl-tRNA Synthetase complexed with phenylalanine has a resolution of 2.7 A and its PDB ID is 1B70.

[0182] Structure databases or Molecular Modeling DataBase (MMDB) contain experimental data from crystallographic structure and NMR assays. Data for MMDB is obtained from Protein Data Bank (PDB). NCBI (National Center for Biotechnology Information) cross-linked structural data to bibliographic information, to sequence databases and NCBI taxonomy. Cn3D, the NCBI 3D Structure Viewer, can be used for easy interactive visualization of molecular structures with Entrez.

[0183] The Entrz 3D Domains database contains protein domains from the NCBI Conserved Domain Database (CDD). Computational biologists determine conserved domains based on sequence patterns or repetitive motifs. The CDD currently contains domains from two popular collections, Smart and Pfam, along with contributions from colleagues at NCBI, like COG. Source databases also provide descriptions and references to citations. Because conserved domains correspond to compact structural units, the CDs contain as far as possible connections to 3D structures via Cn3D.

[0184] To identify conserved domains in a protein sequence, CD-Search support uses a BLAST algorithm specific to the reverse position. The query sequence is compared to a position specific scoring matrix prepared from the basic match of the saved domain. Hits can be displayed as matching pairs of query sequences with a representative domain sequence, or as multiple matches. CD-Search now runs by default in parallel with BLAST protein searches. While the user is waiting in the BLAST queue for further processing of the query, the query domain architecture may already be tested. In addition, CD ART, Conserved Domain Architecture Retrieval Tool allows the user to look for a protein with a similar domain architecture. CD ART uses pre-calculated CD-search results to quickly identify a protein with a set of query-like domains. For more information, see Marchler-Bauer et al., Nucleic Acids

Research 31: 383-387, 2003; and Marchler-Bauer et al., Nucleic Acids Research 30: 281-283, 2002.

[0185] In addition, a database of known aminoacyl-tRNA synthetases has been published by Maciej Szymański, Marzanna A. Deniziak and Jan Barciszewski, in Nucleic Acids Res. 29: 288290, 2001 (titled "Aminoacyl-tRNA synthetases database"). The corresponding website (rose.man.poznan.pl/aars/seq_main.html) contains detailed information about all known AARS from various species. For example, according to the database, Isoleucyl-tRNA Synthetase for radiation-resistant Deinococcus radiodurans (Accession No. AAF10907) contains 1078 amino acids and was published by White et al. In Science 286: 1571-1577 (1999); Walyl-tRNA synthetase for mice (Mus musculus) has 1263 amino acids (Accession No. AAD26531), and was published by Snoek M. and van Vugt H. in Immunogenetics 49: 468-470 (1999); and phenylalanyl-tRNA synthetase sequences for human, Drosophila, S. pombe, S. cerevisiae, Candida albicans, E. coli, and many other bacteria, including Thermus aquaticus ssp. thermophilus, are also available. The database was last updated in November 2006. Similar information can be obtained for other newly identified AARS, for example, by performing a BLAST search using any of the known sequences in the AARS database as a query based on publicly available (such as non-redundant databases at NCBI or "#") or proprietary private databases.

[0186] Alternatively, in some embodiments, if the exact crystal structure for a particular AARS is not known, but its protein sequence is similar or homologous to the known AARS sequence with a known crystal structure. In this case, it is anticipated that the AARS conformation in question is similar to the known AARS crystalline homologous structure. The known structure can therefore be used as the structure for the AARS of interest, or more preferably, it can be used to predict the AARS structure of interest (i.e. in "homology modeling" or "molecular modeling"). As a specific example, the Molecular Modeling Database (MMDB) described above (see, Wang et al., Nuci. Acids Res. 2000, 28: 243-245; Marchler-Bauer et al., Nuci. Acids Res. 1999,27: 240243) provides a search engine that can be used to identify proteins and / or nucleic acids that are similar or homologous to protein sequences (referred to as "adjacent" sequences in MMDB), including adjacent sequences whose three-dimensional structures are known. The database also provides links to known structures, and matching and visualization tools such as Cn3D (developed by NCBI), RasMol, etc., where parent and homologous sequences can be compared, and the structure can be obtained for the parent sequence based on such sequence alignment and known structures.

[0187] A homologous AARS sequence with known 3D structure is preferably at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95% identical, or at least at least about 98% identical to the AARS of interest in the active site region or region

48 knots for binding the amino acid substrate. Such an active site or pocket site cannot be contiguous in the AARS primary amino acid sequence because distant amino acids can meet in 3D structure. In this case, sequence identity or homology can be calculated using, for example, NCBI standard BLASTp programs for standard protein conditions using, in regions adapted together (without insertions or deletions in any of the two compared sequences) and including residues known to play a role in binding the amino acid substrate. For example, the catalytic (alpha) subunit of phenylalanyl-tRNA synthetase from Thermus Aquaticus appears to have an "inserted" region from residues 156 to 165 relative to its homologs from other species. This region can be considered when calculating sequence identity. Alternatively, homologous AARS is preferably about 35%, or 40%, or 45%, or 50%, or 55% identical in general to the AARS of interest. The alpha subunit of phenylalanyl-tRNA synthetase is about 45% total identity and about 80% identity in the active site region to Thermus Aquaticus phenylalanyl-tRNA synthetase. Human alpha subunits of phenylalanyl-tRNA synthetase have about 62%, 60%, 54%, 50% total identity to its counterparts Drosophila, helminth (C. elegans), plant (Arabidopsis thaliana), and yeast (S. cerevisiae), respectively.

[0188] In several cases, when the structure for a particular AARS sequence may not be known or available, it is possible to determine the structure using routine experimental techniques (e.g., X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy) and without undue experimentation. See, e.g., NMR of Macromolecules: A Practical Approach, GCK Roberts, Ed. "Oxford University Press Inc., New York (1993); Ishima i Torchia, Nat. Struci. Biol. 7: 740-743, 2000; Gardner and Kay, Annu. Rev. Bioph. Biome. 27: 357-406, 1998; Kay, Biochem. Cell. Biol. 75: 1-15, 1997; Dayie et al., Annu. Rev. Phys. Chem. 47: 243-282, 1996; Wuthrich, Acta Cyrstallogr. D 51: 249-270, 1995; Kahn et al., J. Synchrotron Radiat. 7: 131-138, 2000; Oakley and Wilce, Clin. Exp. Pharmacol. P. 27: 145-151, 2000; Fourme et al., J. Synchrotron Radiat. 6: 834-844, 1999.

[0189] Alternatively, the three-dimensional structure of the AARS sequence can be calculated from the sequence itself and ab initio using molecular modeling techniques already known in the art. See, e.g., Smith et al., J. Comput. Biol. 4: 217-225, 1997; Eisenhaber et al., Proteins 24: 169-179, 1996; Bohm, Biophys Chem. 59: 1-32, 1996; Fetrow and Bryant, BioTechnol. 11: 479-484, 1993; Swindells and Thorton, Curr. Opin. Biotech. 2: 512-519, 1991; Levitt et al., Annu. Rev. Biochem. 66: 549-579, 1997; Eisenhaber et al., Crit. Rev. Biochem. Moth. 30: 1-94, 1995; Xia et al., J. Mol. Biol. 300: 171-185, 2000; Jones, Curr. Opin. Poison. Biol. 10: 371-379, 2000. Three-dimensional structures obtained from ab initio modeling are usually less reliable than structures obtained by experimental techniques (e.g. NMR spectroscopy or X-ray crystallography) or semi-probable (e.g. homology modeling). However, such structures will typically be of sufficient quality, although less preferably, for use in some of the methods disclosed herein.

-49B. 3D Structure Prediction Methods Based on Sequence Homology [0190] For AARS proteins that have not been crystallized or have been subjected to other assays, a computer generated AARS molecular molecular model and its binding sites can, however, be produced using any of a variety of techniques available in the state. technology. For example, the Ca positions of the target AARS sequence can be assigned to a specific coordinate pattern of the AARS enzyme ("known AARS") having a similar sequence and structure inferred using homology modeling techniques, and the target protein structure and velocity of each atom calculated at simulation temperature (To), at which should be marked with the simulation of amino acid analogue docking. Typically, such a protocol mainly involves predicting the side chain conformation in the target protein of the AARS pattern, accepting a trace of the main chain derived from a tertiary structure, such as predicted by the known AARS protein. Computer programs for performing the energy minimization procedure are often used to generate molecular models. For example, both CHARMM (Brooks et al. (1983) J Comput Chem 4: 187-217) and AMBER (Weiner et al. (1981) J. Comput. Chem. 106: 765) support all molecular system settings, force field calculation and analysis (see also, Eisenfield et al. (1991) Am J Physiol 261: C376-386; Lybrand (1991) J Pharm Belg 46: 49-54; Froimowitz ( 1990) Biotechniques 8: 640-644; Burbam et al. (1990) Proteins 7: 99-111; Pedersen (1985) Environ Health Perspect 61: 185-190; and Kini et al. (1991) J Biomol Struct Dyn 9: 475-488). At the heart of these programs is a set of subroutines that, taking into account the location of each atom in the model, calculate the total potential energy of the system and forces on each atom. These programs can use an initial set of atomic coordinates, parameters for various conditions of potential energy function and a description of the molecular structure topology (covalent). Common features of these molecular modeling methods include: provisions for handling hydrogen bonds and other bonding forces; application of cyclic boundary conditions; and provisions for the occasional adjustment of position, speed or other parameters to maintain or change temperature, pressure, duress or other controlled external conditions.

[0191] Most conventional energy minimization methods use input coordinate data and the fact that the potential energy function is a clear, differential function of Cartesian coordinates to calculate the potential energy and its gradient (which gives strength on each atom) for any set of atomic positions. This information can be used to generate a new coordinate set to reduce the total potential energy and by repeating this process over and over to optimize the structure of the molecule in a given set of external conditions. These energy minimization methods are routinely applied to molecules similar to the AARS proteins in question.

[0192] In general, methods to reduce energy can be carried out for a given temperature, Ti, which may differ from the docking simulation temperature, To. After minimizing the energy of the molecule in Ti, the coordinates and velocities of all atoms in the system are calculated. In addition, the normal operating mode of the system is calculated. Experts in this

-50, they will notice that each normal mode is a collective periodic motion, with all parts of the phase motion system in relation to each other, and that the motion of the molecule is the superposition of all usual ways. For a given temperature, the mean square motion amplitude in a given mode is inversely proportional to the effective force constant for that mode. In this regard, low frequency vibration is often dominated by the movement of the molecule.

[0193] After minimizing the energy of the molecular model in Ti, the system is "heated" or "cooled" to the simulation temperature, To, by carrying out the equilibration course, where the atom velocity is scaled in a gradual manner until the desired temperature, To. The system is further equilibrated for a limited time, up to some system properties such as average kinetic energy. The coordinates and velocities of each atom are obtained from a balanced system.

[0194] Further procedures to reduce energy may also be performed. For example, the second class of methods includes calculating approximate solutions of limited EOM for a protein. These methods use multi-step approaches to solution for Lagrange multipliers and usually only a few repetitions are needed if small corrections are required. The most popular method of this type, SHAKE (Ryckaert et al. (1977) J. Comput. Phys. 23: 327; and Van Gunsteren et al. (1977) Mol. Phys. 34: 1311) is easy to implement and scales as O (N) depending on growth restrictions. Accordingly, the method relates to macromolecules such as AARS proteins. An alternative method, RATTLE (Anderson (1983) J. Comput. Phys. 52:24) is based on the Verlet algorithm speed version. Like SHAKE, RATTLE is an iterative algorithm and can be used to minimize the energy of the AARS protein model in question.

C. Alternative Methods [0195] In other embodiments, instead of retaining the constant identity of the amino acid analog and different AARS structure (by modeling several different mutant structures), the subject method is carried out using molecular model (s) for individually modified AARS (e.g. (in which one more of the non-anchoring amino acid residues are altered) and sampling of many different amino acid analogs or potential fragments to identify analogs that may interact with or be substrates for the modified AARS enzyme. This approach can use amino acid analog coordinate libraries (including rotamer variants) or functional group and spacer libraries that can be linked together to form an amino acid analog side chain.

[0196] Using methods such as those described above, e.g., homologous modeling, a set of coordinates for a binding site for a modified AARS can be derived.

[0197] There are many computational methods that can be easily adapted to identify the structure of amino acid analogs that have adequate steric and electronic properties to interact with the binding site of a modified AARS substrate. See, for example, Cohen et al. (1990) J. Med. Cam. 33: 883-894; Kuntz et al. (1982) J.

-51Mol. Biol 161: 269-288; DesJarlais (1988) J. Med. Cam. 31: 722-729; Bartlett et al. (1989) (Spec. Publ., Roy. Soc. Chem.) 78: 182-196; Goodford et al. (1985) J. Med. Cam. 28: 849857; DesJarlais et al. J. Med. Cam. 29: 2149-2153). Direct methods generally fall into two categories: (1) design by analogy in which the 3-D structures of known molecules (as from crystallographic databases) are docked to the structure of the AARS binding site and are scored for good fit; and (2) de novo design in which the amino acid analogue model is constructed piece by piece at the AARS binding site. The second solution, in particular, may facilitate the development of new molecules uniquely designed to bind the binding site of the modified AARS.

[0198] In an exemplary embodiment, the design of potential amino acid analogs that can act with a particular modified AARS begins with a general point of view of the shape included for the enzyme substrate binding site and a search algorithm capable of scanning a base of fine molecules with known three-dimensional structure for candidates is used geometrically matched to the substrate binding site. Such libraries may generally be small molecule libraries or may be directed to amino acid analogs or small molecules that can be used to create amino acid analogs. It is not expected that the molecules found in the shape search will necessarily be lead structures, as no chemical interaction is evaluated during the initial search. On the contrary, it is envisaged that such candidates may act as a framework for further design, providing molecular skeletons into which appropriate atom substitutes can be inserted. Of course, the chemical contribution of these molecules can be assessed, but the types of atoms are expected to change to maximize electrostatic interactions, hydrogen bond formation and hydrophobic binding to the substrate binding site. Most of these algorithms provide a way to find a wide range of chemical structures that can be complementary to the shape of the AARS binding site substrate.

[0199] For example, each set of small molecules from a specific database, such as the Cambridge Crystallographic Data Bank (CCDB) (Allen et al. (1973) J. Chem. Doc. 13: 119), is separately docked to the binding site of the modified AARS in many geometrically acceptable directions using the docking algorithm. In a preferred embodiment, a set of computer algorithms called DOCK can be used to characterize the shape of the curves and cavities that make up the binding site. See, for example, Kuntz et al. (1982) J. Mol. Biol 161: 269-288. The program can also search the base of small molecules for matrices whose shapes are complementary to a specific binding site of modified AARS. Examples of algorithms that can be adapted for this purpose are described in, for example, DesJarlais et al. (1988) J. Med. Chem. 31: 722-729.

[0200] Directions are evaluated for good fit and the best are kept for further analysis using molecular mechanics programs such as AMBER or CHARMM. Such algorithms have previously been successful in finding the various molecules that are

Complemented to the shape of a given receptor or enzyme binding site and has been shown to have a number of attractive features. First, such algorithms can recover the amazing diversity of molecular architecture. Secondly, the best structures, in previous applications for other proteins, showed an impressive complementarity shape on the extended surface. Third, the general approach seems to be very robust in relation to the small uncertainties of the position of the candidate atoms.

[0201] In certain embodiments, the subject method may use the algorithm described by Goodford (1985, J. Med. Chem. 28: 849-857) and Boobbyer et al. (1989, J. Med. Chem. 32: 1083-1094 ). These articles describe a computer program (GRID) that aims to determine regions with high affinity for various chemical groups (called probes) on the surface of the molecule binding site. GRID is thus a tool to suggest modifications of known ligands that can improve binding. It can be expected that some of these sites outlined by GRID as high affinity regions correspond to "pharmacophore patterns" determined by inference from a number of known ligands. As used herein, the pharmacophore standard is the geometric arrangement of the characteristics of the predicted amino acid analogue that appears to be important for binding. Goodsell and Olson (1990, Proteins: Struct. Funct. Genet. 8: 195-202) used the Metropolis (simulated fusion) algorithm to dock one known ligand to the target protein, and their approach can be adapted to identify appropriate amino acid analogs for docking in AARS binding site. This algorithm can allow torsional flexibility in the amino acid side chain and use GRID interaction energy maps as quick lookup tables to calculate approximate interaction energy.

[0202] Still a further embodiment uses a computer algorithm such as CLIX, which searches such databases as CCDB for small molecules that can be oriented at the substrate binding site with AARS in both spatially acceptable and highly probable ways achieving favorable chemical interactions between the candidate molecule and surrounding amino acid residues. This method is based on characterizing the substrate binding site in terms of a set of preferred binding positions for various chemical groups, and then looking for the orientation of candidate molecules that cause maximum spatial convergence of individual chemical groups of the candidate with the team members. The current availability of computer power dictates that computer-assisted search for new ligands replaces the strategy first in breadth. The goal of the strategy first is to gradually reduce the size of the potential candidate's search space by applying increasingly stringent criteria, as opposed to the strategy first the depth in which the most detailed analysis of the first candidate is performed before proceeding to the next candidate. CLIX complies with this strategy in that its binding analysis is residual - it tries to meet the necessary conditions for matching the hindrance and having individual groups in the "correct" places for binding, without imposing a sufficient condition that the beneficial binding interactions

-53 actually occur. A 'short list' of molecules is ranked, in their privileged orientations, which can then be studied in molecule by molecule mode, using computer graphics and more advanced molecular modeling techniques. CLIX is also able to suggest changes in candidate chemical substituent groups that may improve binding capacity. Again, the start library may be for amino acid analogs or molecules that can be used to generate the amino acid analog side chain.

[0203] Algorithmic details of CLIX are described in Lawerence et al. (1992) Proteins 12: 3141, and the CLIX algorithm can be summarized as follows. The GRID program is used to determine the positions of discrete beneficial interactions (called target sites) at the AARS protein binding site for many representative chemical groups. For each ligand of the candidate in the CCDB, exhaustive attempts are made to make a pair of candidate chemical substituents in a spatial sense at the protein binding site with a pair of corresponding favorable interactions proposed by GRID. All possible combinations of pairs of ligand groups and pairs of GRID sites are considered during this procedure. After locating such convergence, the program rotates the candidate's ligand by about two pairs of groups and checks for spatial hindrance and convergence of other candidate atomic groups with the appropriate target sites. Specific candidate / orientation combinations that are good match geometrically at the binding site and show that sufficient convergence of atomic groups with GRID sites is retained.

[0204] In line with the strategy first across, this approach requires simplification of assumptions. The rigid geometry of the protein and small molecule is maintained at all times. Because the rigid geometry of the first approximation is permissible as coordinates with minimized energy of the modified AARS binding site, they describe the minimum energy for the molecule, although local.

[0205] Another implication hidden in CLIX is that the potential ligand, when introduced into the binding site of the modified AARS substrate, does not change the stereochemistry or partial distribution of the protein charge and thus changes the basis on which GRID energy maps were calculated. It should also be emphasized that the impact sites predicted by GRID are used in the sense of positional type, i.e. when the candidate atomic group is placed in a position predicted to be preferred by GRID, no control is performed to ensure that the binding geometry, protonation state or partial charge distribution promotes strong interaction between the protein and this group. This detailed analysis should be part of a more advanced modeling of candidates selected from the final CLIX list.

[0206] Yet another embodiment of the computer aided molecular design method for identifying amino acid analogs that can be used by a predefined modified AARS involves de novo synthesis of potential inhibitors by algorithmically linking small molecule fragments,

Which will show the desired complementarity with the structural and electrostatic binding site of the enzyme substrate. This methodology uses a large set of small molecule matrices that are iteratively assembled into the AARS substrate binding site model. Each stage of ligand development is assessed according to an energy function based on molecular mechanics that considers van der Waals and Coulomb interactions, internal strain energy of ligand elongation, and desolvation of both ligand and enzyme. The search space can be managed using a data tree controlled by pruning in accordance with applicable criteria.

[0207] In yet another embodiment, potential amino acid analogs can be determined using methods based on a molecular dynamics algorithm quenched by minimizing molecular energy for determining the energy position of preferred functional groups at the binding site of a modified AARS enzyme substrate. This method can help in the construction of molecules that contain this type of functional group by modifying known amino acids as well as amino acid analogues or by de novo synthesis.

[0208] For example, the multiple copy simultaneous search method (MCSS) described by Miranker et al. (1991) Proteins 11: 29-34 may be adapted for use in the subject method. To determine and characterize local functional group minima in the protein force field, multiple copies of selected functional groups are first distributed at the binding site of interest on the AARS protein. Minimizing energy from these copies using molecular mechanics or suppressed dynamics gives clear local minima. The vicinity of these minima can then be examined by grid search or limited minimization. In one embodiment, the MCSS method uses the classic Hartee time-dependent approximation (TDH) to simultaneously minimize or quench multiple identical groups in the protein force field.

[0209] The implementation of the MCSS algorithm requires a selection of functional groups and a molecular mechanics model for each of them. The groups must be simple enough so that they can be easily determined and manipulated (3-6 atoms, little or no dihedral degrees of freedom), but complex enough to approximate the spatial and electrostatic interaction that the functional group would have in the binding substrate to the AARS protein site. A preferred kit is, for example, one in which most organic molecules can be described as a collection of such groups (Patai's Guide to the Chemistry of Functional Groups, ed. S. Patai (New York: John Wiley, and Sons, (1989)). includes fragments such as acetonitrile, methanol, methyl acetate, ammonium, dimethyl ether, methane and acetaldehyde.

[0210] Determining local energy minima at the binding site requires sampling of multiple starting positions. This can be achieved by splitting, for example, 1,0005,000 groups randomly inside the ball centrally at the binding site; only space not occupied by protein should be considered. If the interaction energy of a particular group at a certain location in the protein is more positive than the given cutoff (e.g. 5.0 kcal / mol) the group is removed from this place. Considering the set of starting positions, all

Fragments are simultaneously minimized by using the TDH approximation (Elber et al. (1990) J. Am. Chem. Soc. 112: 9161-9175). In this way, the forces for each episode consist of internal forces and those for protein. An essential element of this method is that the interaction between the fragments is omitted and the forces on the protein are normalized to those from a single fragment. In this way it is possible to perform simultaneous minimization or dynamics of any number of functional groups in the area of ​​one protein.

[0211] Minimization is performed sequentially on subgroups, e.g., 100 randomly placed groups. After a certain number of step intervals, for example 1000 intervals, the results can be tested to remove groups converging to the same minimum. This process is repeated until minimization is complete (e.g. RMS gradient 0.01 kcal / mol / A). Thus, the resulting energy-minimized set of molecules includes what amounts to the set of unconnected fragments in three dimensions representing potential side chains for amino acid analogs.

[0212] The next step is to combine elements with spacers assembled from small chemical units (carbon, chains, or ring moieties) to form amino acid analogues, e.g. each of the detached may be combined in space to produce one molecule using computer programs such , for example NEWLEAD (Tschinke et al. (1993) J. Med. Chem. 36: 3863.3870). The procedure adopted by NEWLEAD executes the following sequence of commands (1) connect two individual groups, (2) keep intermediate solutions for further processing, (3) repeat the above steps for each of the intermediate solutions until no disconnected units are found, and (4) specify at the end of the final solutions, each of which is a single molecule. Such a program may use, for example, three types of spacers: library spacers, single atom spacers, and condensed ring spacers. Library spacers are optimized structures of small molecules such as ethylene, benzene, methylamide. The output generated by programs such as NEWLEAD consists of a set of original molecules containing fragments currently joined by spacers. Atoms belonging to the entrance fragments maintain their original directions in space. Particles are chemically likely due to their simple structure with spacers and functional groups and energetically acceptable due to the rejection of solutions from van der Waals ray breaking.

[0213] Furthermore, the order in which the steps of this method are carried out is merely illustrative. In fact, the steps may be performed in any order or simultaneously, unless otherwise indicated in the present invention.

[0214] Furthermore, the methods disclosed herein can be carried out in any equipment, software, or any combination thereof, since these terms are currently known in the art. In particular, the method of the present invention may be carried out by software, firmware or microcode operating on a computer or computers of any kind. In addition, the software can

-56 contain computer instructions, in any form (e.g. source code, object code, interpreted code, etc.) stored on any computer-readable medium (e.g. ROM, RAM, magnetic media, perforated cards, tape or compact disc (CD) in any form, DVD, etc.). In addition, such software may also be in the form of a computer data signal placed on a carrier wave such as that found on known websites transmitted between devices connected to the Internet. Accordingly, certain embodiments are not limited to any particular platform unless expressly indicated otherwise.

[0215] Exemplary computer hardware means for carrying out certain embodiments may be a Silicon Graphics Power Challenge server with R10000 parallel processors. Suitable software development environment includes CERIUS2 from Biosym / Molecular Simulations (San Diego, CA) or other equivalents.

[0216] The computational methods described above have been effectively used to modify an enzyme synthesis machine (e.g., AARS) to allow the introduction of unnatural amino acids. The same set of calculation tools can also be used to design end products (e.g. monoclonal antibodies and other therapeutic agents) in which unnatural amino acids would be introduced to strengthen or change their structural and functional properties.

[0217] As long as the specific embodiments disclosed herein are described and described, it will be apparent to those skilled in the art that changes and modifications may be made without departing from the broader aspects, and therefore the appended claims are intended to cover all such changes and modifications which fall within the true spirit of the present invention.

Adoption of AARS from various organisms [0218] A second strategy for generating mutated external tRNA, modified or external RS, or modified tRNA / RS pair involves importing tRNA and / or synthetase from another organism into the translational system of interest, such as Escherichia coli. In this particular example, the properties of the heterologous synthetase candidate include, e.g. those that do not attach to the Escherichia coli tRNA rather well (preferably at all) and the properties of the heterologous tRNA candidate include, e.g., those that are not acylated by a Escherichia coli synthetase to a reasonable (preferably not at all).

[0219] Schimmel et al. Reported that GlnRS Escherichia coli (EcGlnRS) does not acylate tRNAGln Saccharomyces cerevisiae (EcGlnRS does not have the N-terminal RNA binding domain owned by GlnRS Saccharomyces cerevisiae (ScGlnRS)). See, EF Whelihan and P. Schimmel, EMBO J., 16: 2968 (1997). For example, the suppressor amber tRNAGln Saccharomyces cerevisiae (SctRNAGlnCUA) was analyzed to determine whether it is also a substrate for EcGlnRS. In vitro aminoacylation tests have shown that this is the case; and in vitro suppression studies show that SctRNAGlnCUA is appropriate in

-57translacji. See, e.g., Liu and Schultz, PNAS. USA, 96: 4780 (1999). ScGlnRS has also been shown not to acylate any Escherichia coli tRNA, only SctRNAGlnCUA in vitro. The extent to which ScGlnRS can aminoacylate SctRNAGlnCUA in Escherichia coli was also assessed using an in vivo complement assay. The non-sensible amber mutation was introduced on a permissive site in the β-lactamase gene. Suppression by the amber mutation of the suppressor tRNA should give full-length β-lactamase and confer ampicillin resistance to cells. When only SctRNAGlnCUA is expressed, the cells show an IC50 of 20 pg / mL ampicillin, indicating virtually no acylation by Escogenichia coli 'endogenous synthetases, when SctRNAGlnCUA is coexpressed with ScGlnRS, the cells gain an IC50 of about 500 pg / mL ampicillin, showing that SccNN aclAl ACl effective in Escherichia coli. See, Liu and Schultz, PNAS, USA, 96: 4780 (1999).

[0220] As another example, tRNA^Asp Saccharomyces cerevisiae is known as an external mutant to Escherichia coli synthetases. See, e.g., Doctor and Mudd, J. Biol. Chem., 238: 3677 (1963); and, Kwok and Wong, Can. J. Biochem., 58: 213 (1980). It was shown that the suppressor amber tRNA derived from it (SctRNA^AspcuA) is also externally mutated in Escherichia coli using the β-lactamase assay described above in vivo. However, the tRNA anti-codon^Asp is a critical element in the diagnosis of AspRS, see, e.g., Giege, et al., Biochimie, 78: 605 (1996), and the anti-codon mutation to CUA leads to loss of suppressor affinity for AspRS. The Escherichia coli E93K AspRS mutant has been shown to recognize the suppressor amber tRNA^AspcuA Escherichia coli an order of magnitude better than wt AspRS. See, e.g., Martin, 'Thesis', Universite Louis Pasteur, Strasbourg, France, 1995. It has been speculated that the introduction of a related mutation in Saccharomyces cerevisiae AspRS (E188K) may restore the affinity for SetRNA^AspCuA · The AspRS mutant (E188K) of Saccharomyces cerevisiae has been found not to acylate Escherichia coli tRNA but to load SctRNA^AspcuA in moderate yield as shown by in vitro aminoacylation experiments. See, e.g., Pastmak, et al., Helv. Chim. Acta, 83: 2277 (2000).

[0221] A similar approach is to use a heterologous synthetase as an external mutated synthetase and mutated initiator tRNA from the same organism or a related organism as the modified tRNA. RajBhandary et al. Found that the human tRNA initiator amber mutant^FMCT is acylated by GlnRS Escherichia coli and acts as an amber suppressor in yeast cells only when EcGlnRS is co-expressed. See, Kowal, et al., PNAS USA, 98: 2268 (2001). This pair therefore represents the outer mutant pair for use in yeast. It was also found that the mutant tRNA initiator amber^tN1et Escherichia coli is inactive towards any Escherichia coli synthetase. A mutant yeast TyrRS was selected that attaches to this mutant tRNA, resulting in an external mutated pair in Escherichia coli.

[0222] Using the methods disclosed herein, the pairs and parts of the pair desired above have evolved to generate mutated external tRNA and / or RS that have the desired characteristics, e.g., which can advantageously aminoacylate Ο-tRNA with an unnatural amino acid.

[0223] In certain embodiments, modified tRNA and modified RS can be obtained by mutating naturally occurring tRNA and RS from various organisms. In one embodiment, the modified tRNA and / or modified RS are from at least one organism, wherein the organism is a prokaryotic organism, e.g., Methanococcus jannaschii, Methanobacterium thennoautotrophicum, Halobacterium, Escherichia coli, A. fulgidus, P. furiosus, P. horikoshii, A. pernix, T. thermophilus or similar. Alternatively, the organism is a eukaryotic organism, e.g. plants (e.g. compound plants, such as monocotyledonous or dicotyledonous), algae, fungi (e.g. yeast, etc.), animals (e.g. mammals, insects, arthropods, etc.), insects, protists or similar. Optionally, the modified tRNA is derived from a mutation of the naturally occurring tRNA from the first organism and the modified RS is derived from a naturally occurring mutation of RS from the second organism. In one embodiment, the modified tRNA and the modified RS may be from mutated tRNA and mutated RS. In certain embodiments, the modified RS and / or modified tRNA from the first organism is delivered to the translational system of the second organism, which optionally comprises endogenous non-functional RS and / or tRNA relative to the codons recognized by the modified tRNA or modified RS.

[0224] The mutated external tRNA and / or the external mutated tRNA synthetase can also optionally be isolated from various organisms. In one embodiment, the mutated external tRNA and / or external mutated synthetase are isolated from at least one organism, wherein the organism is a prokaryotic organism, e.g., Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli, A. fulgidus, P. furiosus, P. horikoshii, A. pernix, T. thermophilus, or similar. Alternatively, the organism is a eukaryotic organism, e.g. plants (e.g. compound plants, such as monocotyledonous or dicotyledonous), algae, fungi (e.g. yeast, etc.), animals (e.g. mammals, insects, arthropods, etc.), insects, protists or similar. Optionally, the external tRNA is isolated from the naturally occurring tRNA from the first organism and the external mutant synthetase is isolated from the naturally occurring RS from the second organism. In one embodiment, the mutated external tRNA and / or external mutated tRNA synthetase can be isolated from one or more libraries (which optionally contain one or more tRNA and / or RS from one or more organisms (including those including prokaryotes and / or eukaryotes).) [0225] Also disclosed herein are methods for selecting a pair of mutant external tRNA and / or tRNA synthetase for use in any translation system. These methods include: introducing a marker gene, tRNA and / or aminoacyl-tRNA synthetase (RS) isolated or derived from the first organism into the first set of cells from the second organism; insertion of a marker gene and tRNA or RS into a duplicate set of cells from a second organism; and, selecting for surviving cells in the first set that do not survive in the duplicate set of cells, wherein the first set and duplicate of the set of cells are cultured in the presence of a selective agent, and wherein the surviving cells contain the mutated external tRNA and / or RS to be used in the translation system.

In one embodiment, the comparison and selection includes an in vivo complement assay. In another embodiment, the concentration of the selection agent is varied. The same test can also be carried out in vitro or in vivo based on a second organism.

AARS Generation by Mutagenesis and Selection / Screening [0226] AARS mutation or modification for use to introduce an unnatural amino acid into a target polypeptide or protein can be performed by site-directed mutagenesis after identifying the desired contact amino acid residues. Identification of contact amino acids can be carried out by any method that allows analysis of the AARS structure, including crystallographic analysis, computer modeling, nuclear magnetic resonance (NMR) spectroscopy, library screening, or a combination of any of these or other methods.

[0227] Many AARS molecules have been sequenced and they provide guidance as to which amino acids are important for binding the amino acid on which the appropriate tRNA can be loaded. See, for example, SEQ ID Nry. 48-103.

[0228] In certain embodiments, AARS capable of attaching to a specific mutant external tRNA of a particular unnatural amino acid can be obtained by AARS mutagenesis to generate a library of candidates, followed by screening and / or selection of an AARS candidate allowing their desired function. Such external mutated AARS and mutated external tRNA can be used to produce in vitro / in vivo the desired protein with modified unnatural amino acids.

[0229] Thus, methods for producing protein biosynthesis machine components, such as mutated external RS, mutated external tRNA, and / or mutated external tRNA / RS pairs that can be used to introduce an unnatural amino acid are provided in some of the embodiments disclosed herein.

[0230] In one embodiment, methods for producing at least one recombinant external mutant aminoacyl-tRNA synthetase include: (a) creating a library (optionally mutated) RS derived from at least one aminoacyl-tRNA synthetase (RS) from a first organism, e.g. a eukaryotic organism (such as yeast), or a prokaryotic organism such as Methanococcus jannaschii, Methanobacterium thennoautotrophicum, Halobacterium, Escherichia coli, A. fulgidus, P. furiosus, P. horikoshii, A. pemix, T. thermophilus, or the like; (b) selecting (and / or screening) the RS library (optionally mutated RS) for members who aminoacylate the mutated external tRNA in the presence of an unnatural amino acid and a natural amino acid, thereby providing a pool of active (optionally mutated) RS; and / or (c) selecting (optionally by negative selection) the pool for active RS (e.g. mutant RS) that preferentially aminoacylate Ο-tRNA in the absence of an unnatural amino acid, thereby providing at least one

A recombinant outer mutant synthetase, wherein at least one recombinant outer mutant synthetase preferentially aminoacylates the mutated outer tRNA with an unnatural amino acid. Recombinant external mutant synthetases produced by these methods are also disclosed in certain embodiments disclosed herein.

[0231] In one embodiment, the RS is an inactive RS that can be generated from mutating the active RS. For example, an inactive RS may be generated by mutating at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, or at least about 10 or more amino acids to different amino acids e.g. alanine.

[0232] Mutant RS libraries can be generated using various mutagenesis techniques known in the art. For example, mutated RSs can be generated by site-specific mutations, random mutations, diversity recombinant mutations, chimeric constructs, and other methods described herein or known in the art.

[0233] In one embodiment, selecting (and / or screening) an RS library (or mutant RS) for members who are active, e.g., who aminoacylate the mutated external tRNA in the presence of an unnatural amino acid and a natural amino acid, includes: introduction of a positive selection marker or a screening marker, e.g. an antibiotic resistance gene, or similar, and a library (optionally mutated) of RS into multiple cells, wherein the positive selection marker and / or screening marker contains at least one codon whose translation (optionally conditionally) depends on the ability of candidate RS to attach to mutated external tRNA (natural and / or unnatural amino acids); culturing multiple cells in the presence of a selective agent; identification of surviving cells (and showing a specific response) in the presence of a selective agent and / or for translational screening with a successful codon in a positive selection marker or for screening, thus providing a subset of positively selected cells that contain a pool of active (or mutated) RS. Optionally, the concentration of the selection and / or screening agent can be changed. In certain embodiments, the cells do not contain functional endogenous tRNA, RS or tRNA-RS pair, which may help translate the codon. Endogenous tRNA / RS pair can be turned off by gene deletion and / or RS inhibitors.

[0234] Because many important cell genes may also contain a codon that depends on the external mutant synthetase's ability to load a modified tRNA in the absence of a functional endogenous RS / tRNA pair, in one embodiment, no positive selection markers are required for the positive selection process - cell survival can be used to read the positive selection process.

[0235] In one aspect, the positive selection marker is the chloramphenicol acetyltransferase (CAT) gene. Optionally, the positive selection marker is the β-lactamase gene. In another aspect, a positive screening marker includes a fluorescent or luminescent screening marker or an affinity based screening marker (e.g., cell surface marker).

[0236] In a similar embodiment, the cell-free in vitro system can be used to test the external ability of a mutated synthetase to load a modified tRNA in a positive screening test. For example, the ability of an in vitro system to translate a positive screening gene, such as a fluorescent marker gene, may depend on the external mutant synthetase's ability to load a modified tRNA to read the codon of the marker gene.

[0237] In one embodiment, negative pool selection or screening for active RS (optionally mutated) that preferentially aminoacylate the mutated tRNA in the absence of an unnatural amino acid includes: introducing a negative selection marker or screening marker for a pool of active (or mutated) RS from selection or positive screening for multiple translation systems, wherein the negative selection marker or screening marker comprises at least one codon (e.g. codon for a toxic marker gene, e.g., the ribonuclease bamase gene), whose translation depends on the ability of the RS candidate to load the mutated external tRNA (natural amino acid); and, identifying a translation system that shows a specific response in screening in the first medium supplemented with unnatural amino acid and screening or selection agent, but does not show a specific response in the second medium supplemented with natural amino acid and selection or screening agent, thereby providing surviving cells or cells screened with at least one recombinant RS.

[0238] In one aspect, the concentration of selection agent (and / or screening) varies. In some aspects, the first and second organisms are different. In this way, the first and / or second organism optionally includes: prokaryotes, eukaryotes, mammals, Escherichia coli, fungus, yeast, archaebacterium, eubacterium, plant, insect, protist, etc. In other embodiments, the screening marker comprises a fluorescent or luminescent screening marker (like green fluorescent protein) or an affinity based screening marker.

[0239] In addition, some aspects include the situation where the negative selection marker includes the ribonuclease bamase gene (which contains at least one said codon). Other aspects include the situation in which the screening marker optionally comprises a fluorescent or luminescent screening marker or an affinity based screening marker. In the embodiments herein, screening and / or selection and optionally include a variation of screening and / or selection stringency.

[0240] In one aspect, a second set of mutated RSs derived from at least one recombinant RS can be generated by mutagenesis, e.g., random mutagenesis, site-specific mutagenesis, recombination or a combination thereof.

[0241] The methods herein optionally comprise a situation in which the unnatural amino acid is selected from, e.g. O-methyl-L-tyrosine, L-3- (2-naphthyl) alanine, 3-methyl-phenylalanine, O4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcP-serine , L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonothyrosine, piodine-phenyl bromophenylalanine, p-amino-L-phenylalanine, and isopropyl L-phenylalanine. Recombinant RS prepared by the methods disclosed herein are also included in the embodiments disclosed herein.

[0242] In a related aspect, methods for producing a recombinant mutant external tRNA include: (a) creating a library of mutated tRNA derived from at least one tRNA from the first organism; (b) selection (e.g. negative selection) or screening the library for (optionally mutated) tRNAs that are aminoacylated by aminoacyl-tRNA synthetase (RS) from the second organism in the absence of RS from the first organism, thereby providing a pool of tRNA (optionally mutated); and, (c) selecting or screening the tRNA pool (optionally mutated) for members who are aminoacylated by the introduced external mutant RS, thereby providing at least one recombinant tRNA; wherein at least one recombinant tRNA recognizes the codon of unnatural amino acids and is not effectively recognized by RS from the second organism and is preferably aminoacylated by an external mutated RS.

[0243] The various methods disclosed herein optionally include a situation in which selection or screening involves one or more positive or negative selections or screenings, e.g., change in amino acid permeability, change in translation efficiency, and change in translational fidelity. In addition, one or more lesions are optionally based on a mutation of one or more genes in the body in which the mutated external tRNA-tRNA synthetase pair is used to produce such proteins. Here, selection and / or screening optionally include situations where at least 2 codons are used in one or more selection genes or in one or more genes for screening. Such multiple codons are optionally in the same gene or in different selection / screening genes. In addition, optional multiple codons are optionally different codons or contain the same type of codons.

[0244] Kits are an additional feature of certain embodiments disclosed herein. For example, kits may contain one or more translation systems as set forth above (e.g., a cell), one or more tRNA (including modified or mutated tRNAs), one or more AARS (including modified or mutated AARS), one or more unnatural amino acids, e.g. with suitable packaging material, containers for holding components in set, instructional materials for implementing the methods disclosed herein, and / or

-63podobne. If one or more AARS and / or one or more tRNA are provided in the kit, they may be provided as nucleic acids or proteins and may be part of one vector or contained in separate vectors. Similarly, translation system products (e.g., proteins such as EPO analogues containing unnatural amino acids) can be provided in kit form, e.g. with containers for holding components in the kit, instructional materials for implementation in the methods disclosed herein, and / or the like.

Application examples [0245] More than 100 uncoded amino acids (all ribosomally acceptable) have been reported to be introduced into proteins using other methods (See, for example, Schultz et al., J. Am. Chem. Soc., 103: 1563-1567, 1981; Hinsberg et al., J. Am. Chem. Soc., 104: 766-773, 1982; Pollack et al., Science, 242: 1038-1040, 1988; Nowak et al., Science, 268: 439-442, 1995) all of these analogues can be used in the subject method to efficiently incorporate such protein analogues into products.

[0246] In another preferred embodiment, two or more analogs can be used in the same translation system in vitro or in vivo, each with its mutated external tRNA or a pair of external mutated synthetases. This is more easily achieved when the natural amino acid is encoded by four or more codons (like six for Leu and Arg). However, for amino acids encoded by only two codons, one can be reserved for the natural amino acid, while the other can be "shared" by one or more amino acid analogues. These analogues may resemble only one natural amino acid (e.g., different Phe analogs), or resemble other amino acids (e.g., Phe and Tyr analogs).

[0247] In certain embodiments, a first nucleic acid encoding an external mutated / modified tRNA molecule that is not efficiently loaded by endogenous aminoacyl-tRNA synthetase in an in vitro (IVT) cell / translation system or the external mutated / modified tRNA alone. In accordance with some embodiments, the second nucleic acid encoding the outer mutated / modified aminoacyl tRNA synthetase (AARS) is introduced into the cell / IVT. The outer mutated / modified AARS is capable of attaching to the outer mutated / modified tRNA of the selected amino acid analog. The amino acid analogue can then be introduced into the cell so that it can be introduced into one or more proteins within the cell or IVT.

[0248] In other embodiments, the environment is a cell. Various cell lysates (or their lysates suitable for IVT) may be used in certain methods, including, for example, a bacterial cell, fungal cell, insect cell, and mammalian cell (e.g., a human cell or an inhuman mammalian cell). In one embodiment, the cell is an E. coli cell, in another embodiment, the cell is a Pseudomonas cell.

[0249] In certain embodiments, an amino acid analog can be obtained by directly contacting a cell or IVT with an analog, for example, using a solution of the analog to the cell in culture, directly or by adding the analog to IVT. An analog can also be provided by introducing one or more additional nucleic acid constructs into a cell / IVT, wherein the additional nucleic acid constructs encode one or more proteins from the synthesis with amino acid analogs that are necessary for the synthesis of the desired analog.

[0250] Certain embodiments further include introducing the nucleic acid construct matrix into the cell / IVT, wherein the matrix encodes the protein, wherein the nucleic acid construct contains at least one sequence of degenerate codons. Nucleic acids introduced into the cell / IVT can be introduced as one construct or multiple constructs. In certain embodiments, different nucleic acids are introduced in the same construct. For example, the nucleic acids can be used in any suitable vectors capable of expressing encoded tRNA and / or proteins in the / IVT cell. In one embodiment, the first and second nucleic acid sequences are provided in one or more plasmids. In another embodiment, the vector or vectors used are viral vectors, including, for example, adenoviral vectors and lentiviruses. These sequences can be inserted into the appropriate cell / IVT promoter sequences, or multiple sequences that can be inducible to control sequence expression.

[0251] For in vitro use, one or more external mutant synthetases can be recombinantly produced and delivered to any available in vitro translation systems (such as commercially available Wheat Germ Lysate based PROTEINscript-PRO ™, E. coli Ambion® system for conjugated transcription / translation in vitro; or based on Retic Lysate IVT ™ Kit rabbit rabbit reticulocyte (Ambion®). Optionally, the in vitro translation system can be selectively depleted in one or more natural AARS (by, for example, immuno-depletion using AARS immunobilized natural antibodies) and / or natural amino acids so that increased analogue introduction can be achieved. Alternatively, nucleic acids encoding the re-engineered external mutant synthetases may be provided in place of the recombinantly produced AARS. Analogs for incorporation into mature protein products are also provided for the in vitro translation system.

[0252] Although in vitro protein synthesis cannot usually be performed on the same scale as in vivo synthesis, in vitro methods can give hundreds of micrograms of purified protein containing amino acid analogs. Such proteins are produced in quantities sufficient for their characterization using circular dichroism (CD), nuclear magnetic resonance spectrometry (NMR), and X-ray crystallography. This method can also be used to study the role of hydrophobicity, packaging, entropy in the side chain and hydrogen bonding in determining protein stability and folding. It can also be used to study the catalytic mechanism, signal transduction and transfer

-65 electrons in proteins. In addition, the properties of proteins can be modified using this method. For example, photoclatted proteins can be generated that can be activated by photolysis and the introduction of new chemical handles into proteins for a specific insertion site for optical and other spectroscopic probes.

[0253] Development of a general approach for introducing amino acid analogues into proteins in vivo, directly from the culture medium, to significantly increase the mutagenesis potency of the unnatural amino acid. For example, the ability to synthesize large amounts of proteins containing heavy atoms will facilitate the determination of the protein structure, and the ability to specifically substitute fluorophores or photo-cleavable groups to proteins in living cells will provide powerful tools to study protein function in vivo. Alternatively, the properties of proteins could be improved by providing building blocks with new functional groups such as ketone containing amino acids.

[0254] For in vivo use, one or more AARS may be delivered to a host cell (prokaryotic or eukaryotic) as biological material, such as coding sequences on plasmids or viral vectors that can optionally be integrated into the host genome and constitutively or inductively expressed redesigned AARS. The heterologous or endogenous protein of interest can be expressed in such a host cell in the presence of provided amino acid analogs. Protein products can then be purified using any of the purification methods known in the art of proteins, or techniques specifically designed for the protein of interest.

[0255] Here are some of the possible ways to generate a transcript that encodes a polypeptide. In general, any means known in the art for producing transcripts can be used to synthesize protein with amino acid analogs. For example, any in vitro transcription system or conjugated transcription / translation systems can be used to generate the transcript of interest that then serves as a template for protein synthesis. Alternatively, any cell, cell / cell line designed by engineering methods, or functional elements (lysates, membrane fractions, etc.) that are capable of expressing a protein from a biological material can be used to generate a transcript. These transcript preparation agents will typically contain components such as RNA polymerase (T7, SP6, etc.) and cofactors, nucleotides (ATP, CTP, GTP, UTP), necessary transcription factors and appropriate buffer conditions as well as at least one suitable DNA template, but other parts can also be added to optimize reaction conditions. One skilled in the art will be able to easily anticipate other embodiments similar to those described herein.

Chemical moieties [0256] In certain embodiments, the unnatural amino acid (s) and / or therapeutic molecule comprise a chemically reactive group. The moiety may be strongly nucleophilic or electrophilic and thus is available for reaction directly with the therapeutic molecule or antibody or fragment thereof. Alternatively,

The grouping may be a weaker electrophile or nucleophile and therefore requires prior activation prior to conjugation with the therapeutic molecule or antibody or fragment thereof. Such an alternative would be desirable when it is necessary to delay the activation of a chemically reactive moiety until the agent is added to the molecule in order to avoid reaction of the agent with the moiety. In another scenario, the moiety is chemically reactive, the scenarios differ (in response to the antibody scenario) depending on whether, after adding the agent, the moiety is directly reacted with the antibody or fragment thereof, or reacted first with one or more chemicals to make the moiety capable of reacting with the antibody or fragment thereof. In certain embodiments, a chemically reactive moiety comprises an amino group, a thiol group, a hydroxyl group, a group containing a carbonyl group, or an alkyl leaving group.

[0257] Certain embodiments may employ Klik chemistry, which includes, but is not limited to, 1,3-dipolama Huisgen cycloaddition, in particular a Cu (I) catalyzed step variant, Diels-Alder reaction, nucleophilic substitution especially for small strained rings such , such as aziridine and epoxy compounds, the formation of ureas and amides similar to carbonyl chemistry, addition reactions to carbon-carbon double bonds such as epoxidation and dihydroxylation.

[0258] Thus, in addition to or instead of glycosylating the polypeptides of the embodiments disclosed herein, other chemical moieties (including poly (ethylene glycol)) can be added, linked, attached or otherwise conjugated or introduced into modified polypeptides. PEGylation is the process of covalent attachment of oligosaccharides and synthetic polymers such as polyethylene glycol (PEG) specifically on therapeutic protein molecules. PEGylation of a protein can significantly increase the half-life, shielding the polypeptide from proteolytic enzymes and increasing the apparent size of the protein, thereby reducing clearance. In addition, PEG conjugates can improve protein solubility and have a beneficial effect on biodistribution. The physical and pharmacological properties of PEGylated proteins depend on the number and size of PEG chains attached to the polypeptide, the location of PEG sites and the chemistry used for PEGylation.

[0259] Examples of PEG conjugation of proteins include the reaction of NEG-hydroxysuccinimidyl esters derivatized with PEG lysine, reactions of 1,4-addition of PEGs derivatized with maleimide and vinylsulfone with cysteine, and condensation with a hydrazide containing PEG with aldehydes produced by glycoprotein oxidation. If there is more than one reactive site in the protein (e.g. multiple amino groups or thiol groups) or reactive electrophiles are used, non-selective attachment of one or more PEG molecules may occur, resulting in a heterogeneous mixture that is difficult to separate. The lack of selectivity and positional control regarding attachment of PEG chains can lead to significant losses of biological activity and possible increased immunogenicity of the conjugated protein. In fact, in the past, loss of biological activity and product heterogeneity were the most common problems associated with the development of long-acting

-67 protein drugs using standard PEGylation techniques. Modification of amino acids with PEG reactive proteins usually results in drastic loss of biological activity due to modification of lysine residues located in regions of the protein essential for biological activity. In certain situations, the biological activity of growth hormones can be reduced 400-fold or more. For example, GCSF bioactivity is reduced 1,000-fold when proteins are modified by conventional amino-PEGylation techniques (Clark et al., J. Biol. Chem. 271: 21969, 1996; Bowen et al., Exp. Hematol. 27, 425, 1999). Thus, there is a need for a method that allows completely site-specific and irreversible attachment of PEG chains to proteins.

[0260] It would be beneficial to use advanced protein engineering technology to create long-acting, "patient-friendly" human protein pharmaceuticals, for example, by introducing unnatural amino acids into the drug protein such that the engineered protein drug can achieve a longer half-life and / or extended or even enhanced biological activity. To this end, certain embodiments disclosed herein can be used to overcome problems such as diversity and loss of activity occurring in standard aminoPEGylation techniques. The incorporation of unnatural amino acids will provide unique, predetermined sites away from the binding site or catalytic site on the target protein, with PEG molecules being specifically conjugated. In addition, PEG molecules can be attached to unnatural amino acids by techniques other than amino-PEGylation, thereby saving primary lysine amino groups from undesired PEGylation. These techniques can be used to increase the half-life, efficacy and / or biological safety of pharmaceutical products in all areas, including specific cancer, endocrinology, infectious diseases and inflammation, etc.

[0261] As an illustrative example, click chemistry or cycloaddition can be used to form a triazole bond. One particular example of cycloaddition is copper-mediated Huisgen [3 + 2] cycloaddition (Tomoe et al., J. Org. Chem. 67: 3057, 2002; Rostovtsev et al., Angew. Chem., Int. Ed. 41: 596, 2002; and Wang et al., J. Am. Chem. Soc. 125: 3192, 2003) azide and alkyne is an external mutant to all functional groups present in proteins and forms a stable triazole binding, this reaction can be used for selective PEGylation of proteins. For example, Deiters et al. (Bioorg. Med. Chem. Lett. 14 (23): 5743-5745, 2004) present a generally applicable PEGylation method based on the introduction of site-specific para-enzyme into phenylalanine into proteins in yeast. The azide group was used in a mild cycloaddition reaction [3 + 2] with an alkyne derivatized PEG reagent to obtain a selectively PEGylated protein. This strategy should be useful for the production of selective PEGylated proteins for therapeutic applications.

] 0262] In certain embodiments, the polypeptide is a therapeutic, diagnostic or other protein selected from: Alpha-1 antitrypsin, Angiostatin, Factor

Antianthytholytic antibodies (including the antibody or functional fragment or derivative thereof selected from: Fab, Fab ', F (ab) 2, Fd, Fv, ScFv, diabody, tricep, tetrabody, dimer, trimer or mini body), vasogenic molecules, angiostatic molecules, apolipoproteins, Apoprotein, Atrial natriuretic factor, Atrial natriuretic polypeptide, Asparaginase Adenosine deaminases, Hirudin, Ciliary neurotrophic factor, bone morphogenetic factor (any and all BMPs), Vestibular peptides, CXC chemokines (e.g. T39765, NAP-2, ENA-78, Gro-a, Gro-b, Groc, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG), Calcitonin, CC chemokines (e.g. Monocyte-1 chemotactic protein, Monocyte-2 chemotactic protein, Monocyte-3 chemotactic protein, Monocyte-1 alpha inflammatory protein, Monocyte-1 beta inflammatory protein, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262), CD ligand , calcitonin, c-kit ligand, collagen, colony growth factor (CSF), C-type natriuretic peptide (CNP), Complement Factor 5a, Complement Inhibitor, Complement Receptor 1, cytokines (e.g. Epithelial Neutrophil Activating peptide-78, GROa / MGSA, GROp, GROy, la-la, MIP-Ιδ, MCP1), deoxyribonucleic acids, Epidermal Growth Factor (EGF), Erythropoietin, Exfoliating toxins A and B, Factor IX, Factor VII, Factor VIII, Factor X, Fibroblast growth factor (FGF), Fibrinogen, Fibronectin, granulocyte colony stimulating factor (G-CSF), granulocyte and macrophage colony stimulating factor (GM-CSF), follitropin, glucocerebrosidase, gonadotropin, glucagon GLPI, growth factors, Hedgehog proteins (e.g. Sonic, Indian, Desert), Human Growth Hormone, Hemoglobin, Hepatocyte Growth Factor (HGF), Hepatitis Viruses, Hirudin, Human Serum Albumin, Insulin, Insulin Growth Factor (IGF), interferons (e.g. IFN-a, IFN-β, IFN-γ, IFN-ε, IFN-ζ, IFN-η, IFN-k, IFN-λ, IFN-τ, IFN5&gt;, IFN-w), interleukins (e.g. IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, ILII, IL-12, IL-13, IL-14, IL-15, etc.), Keratinocyte growth factor (KGF), Lactoferrin, leukemia inhibitory factor, Luciferase, Luteinizing Hormone, Neurturin, Neutrophil inhibitory factor (NIF), Oncostatin M, osteogenic protein, Parathyroid hormone, PDECSF, PDGF, peptide hormones (e.g. Human Growth Hormone), Pleiotropin, Protein A, Protein G, Phenylalanine Hydroxylase, Parathyroid hormone (PTH), Prolactin, Pyrogenic A, B and C Exotoxins, Relaxin, Renin, Ribonucleic Acids, SCF, Soluble Complement Receptor I, Soluble IC AM 1, Soluble interleukin receptors (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL- 13, IL-14, 1IL-5), Soluble TNF receptor, Somatomedin, Somatostatin, Somatotropin, Superantigens, Streptokinase, i.e. Staphylococcal enterotoxins (SEA, SEB, SEC1, SEC2, SEC3, SED, SEE), Superoxide dismutase (SOD), Toxic shock syndrome toxin (TSST-1), Thymosin alpha 1, Tissue plasminogen activator, Tumor necrosis factor (TNF beta) , Tumor Necrosis Factor Receptor (TNFR), Tumor Necrosis Factor Alpha (TNF alpha), Tumor Necrosis Factor Ligand Inducing Apoptosis (TRAIL), Vascular Endothelial Growth Factor (VEGEF), Urokinase; a transcription modulator that modulates growth, differentiation or regulation, where the transcription modulator is derived from prokaryotes, eukaryotes or viruses, fungi, including plants, yeast, insects and animals, including mammals; expression activator selected from cytokines,

Inflammatory molecules, growth factors and their receptors, oncogen products, interleukin (e.g. IL-1, IL-2, IL-8, etc.), interferons, FGF, IGF-I, IGF-II, FGF, PDGF, TNF , TGF-a, TGF-β, EGF, KGF, SCF / c-Kit, CD40L / CD40, VLA-4 / VCAM-1, ICAM-1 / LFA-1, and hyalurin / CD44; signal transduction molecules and related oncogen products, e.g., Mos, Ras, Raf and Met; transcriptional activators and suppressors, e.g. p53, Tat, Fos, Myc, Jun, Myb, Rei; Steroid hormone receptors selected from receptors for estrogen, progesterone, testosterone, aldosterone, LDL or corticosterone; or an enzyme selected from: amidase, amino acid racemase, acylase, dehalogase, dioxygenase, diaryl propane peroxidases, epimerases, epoxy hydrolases, esterases, isomerases, kinases, glucose isomerases, glycosidases, glycosyl transferases, haloperoxidases, e.g. p450s), lipases, lignin peroxidase, nitrile hydrates, nitrilases, proteases, phosphatases, subtilisins, transaminases or nucleases.

[0263] In the event that the protein or molecule of interest to be modified is an antibody or antibody fragment, the unnatural amino acid residue (s) may be located anywhere or position in the antibody structure, depending on the desired target. For example, an unnatural amino acid residue can be placed in a Fab variable region, an Fc region, or other site that interacts with the Fc region of an antibody. In other embodiments, an unnatural amino acid residue may be located in the antibody binding interface or Vh- region In some embodiments, the modified antibody exhibits an increase or decrease in the ability to kill one or more targets. In particular, an antibody with an increased ability to kill one or more targets, or reduced side effects may be desirable.

[0264] In other embodiments, the unnatural amino acid (s) confer increased binding affinity for the Fc receptor and / or for the Clq complement system. In particular, the modified antibody may have altered (e.g., increased) affinity and / or specificity for the antigen or partner binding protein (e.g., C1 q complement and / or Fc receptor on macrophages, etc.). For example, modification of the molecule may increase or decrease antibody dependent cellular cytotoxicity (ADCC) function or complement binding activity. In other examples, modification of a particular molecule may increase or decrease its ability to bind to another molecule structure of a natural counter (e.g., an antibody).

Glycosylation by Unnatural Amino Acids [0265] Post-translational modifications of proteins by glycosylation methods can affect protein folding and stability, modification of protein internal activity, and modulation of their interaction with other biomolecules. See, e.g., Varki, Glycobiology 3: 97-130, 1993. Natural glycoproteins are often present in the population as different sugar forms, making glycan structure analysis and studying the effect of glycosylation of protein structure and function difficult. Thus, methods for the synthesis of natural and unnatural homogeneously glycosylated proteins are needed to systematically understand glycan function and to develop improved glycoprotein therapeutic agents.

[0266] The previously known approach to producing proteins with desired glycosylation patterns uses glycosidases to convert a natural heterogeneous glycoprotein to a simple homogeneous core on which saccharides can be grafted sequentially with glucosyl transferases. See, e.g., Witte et al., J. Am. Chem. Soc. 119: 2114-2118, 1997. A limitation of this method is that the primary glycosylation sites are predetermined by the cell line in which the protein is expressed. Alternatively, the glycopeptide containing the desired glycan structure can be synthesized by solid phase peptide synthesis. This glycopeptide can be combined with other peptides or fragments of recombinant proteins to obtain a larger native glycoprotein (see, e.g., Shin et al., J. Am. Chem. Soc. 121: 11684-11689, 1999), expressed ligation ( see, e.g., Tolbert and Wong, J. Am. Chem. Soc. 122: 5421-5428, 2000) or with engineered protease (see, e.g. Witte et al., J. Am. Chem. Soc. 120: 1979-1989, 1998). Both native chemical ligation and expressed protein ligation are most effective in small proteins and require a cysteine ​​residue at the N-terminus of the glycopeptide.

[0267] When a protease is used for ligation of peptides together, the ligation site may be located away from the glycosylation site for good coupling efficiency. See, e.g., Witte et al., J. Am. Chem. Soc. 120: 1979-1989, 1998. The third approach is to modify the saccharidary protein directly using chemical methods. Good selectivity can be achieved with saccharide haloacetamide derivatives that are linked to the cysteine ​​thiol group (see, e.g., Davis and Flitsch, Tetrahedron Lett. 32: 67936796, 1991; and Macmillan et al., Org. Lett. 4: 1467-1470, 2002). But this method can become problematic for proteins that have more than one cysteine ​​residue.

[0268] Certain embodiments disclosed herein provide methods for glycoprotein synthesis. These methods include, in some embodiments, introducing into the protein an unnatural amino acid that contains a first reactive group; and contacting the protein with a saccharide moiety that contains a second reactive group, wherein the first reactive group reacts with the second reactive group, thereby forming a covalent bond that attaches the saccharide moiety to the unnatural amino acid of the protein. The glycoproteins produced by these methods are also included in some embodiments.

[0269] The first reactive group is, in some embodiments, an electrophilic moiety (e.g., a ketone moiety, aldehyde moiety, and / or the like) and the second reactive group is a nucleophilic moiety. In some embodiments, the first reactive group is a nucleophilic moiety and the second reactive group is an electrophilic moiety (e.g., ketone moiety, aldehyde moiety, and / or the like). For example, an electrophilic moiety is attached to a saccharide moiety and the nucleophilic moiety is attached to an unnatural amino acid. The saccharide moiety may contain a single carbohydrate group or the saccharide group may contain two or more carbohydrate residues.

[0270] In some embodiments, the methods may further include contacting the saccharide moiety with a glycosyl transferase, donor moiety

-Saccharide and other reagents needed for glycosyltransferase to function for a sufficient period of time and under suitable conditions to transfer the saccharide with the saccharide donor moiety to the saccharide moiety. The product of this reaction can, if desired, be contacted via at least a second glycosyltransferase together with a suitable saccharide donor moiety.

[0271] In certain embodiments, the method further comprises contacting the saccharide moiety with one or more of pi-4N-acetylglucosaminyl transferase, al, 3-fucosyl transferase, al, 2-fucosyl transferase, al, 4-fucosyl transferase, βΙ-4-galactosyl transferase , sialyltransferase and / or the like to form a biantenam or triantenam oligosaccharide structure. In one embodiment, the saccharide residue contains a GlcNAc terminal, the saccharide donor moiety is UDP-Gal and the glycosyltransferase is β-1,4-galactosyltransferase.

[0272] In one embodiment, the saccharide moiety comprises a terminal GlcNAc, the saccharide donor moiety is UDP-GlcNAc and the glycosyltransferase is βΐ4N-acetylglucosaminyltransferase.

[0273] Optionally, some methods may further include contacting the product from the N-acetylglucosaminyl transferase reaction with β1-4-manosyltransferase and GDP-mannose to form a saccharide group that contains zawieraηβ1-401εΝΑοβ1-401οΝΑο. Optionally, the method further comprises contacting the Manpi-4GlcNAc31-4GlcNAc moiety with al3mannosyltransferase and GDP-mannose to form a saccharide group that contains Manal3ΐνυηβ1-401οΝΑοβ1-401εΝΑε-. Optionally, the method further comprises contacting the moiety ΐνυηα1-3Μ3ηβ1-4ΟΙεΝΑεβ1-4ΟΙεΝΑε with al-6 mannosyltransferase and GDPmannose to form a saccharide group which contains Γνυηα1-6 (Μ3ηα1-3) Ι ^ ηβ1-401lcNAc14. Optionally, the method further comprises contacting the Manal6 (Ν ^ ηα1-3) Ν ^ ηβ1-401εΝΑοβ1-401εΝΑϋ moiety with βl-2N-acetylglucosaminyltransferase and UDPGlcNAc to form a saccharide group that contains Manal-6 (GlcNAcNA 1-23G-1-Manal 4GlcNAc-. Optionally, the method further comprises contacting the Μ3ηα1-6 (01οΝΑεβ1-2Μ3ηα1-3) Μ3ηβ1-401εΝΑοβ1-401οΝΑε moiety with β1-2Νacetylglucosaminyl transferase and UDP-GlcNAc to form a saccharide group that contains MMΟΕΝΑ β -anaβ 3) Μ3ηβ 1 -4Ο1εΝΑεβ 1 -4GlcN Ac-.

[0274] The step of introducing an unnatural amino acid into the protein that contains the first reactive group, in some embodiments, involves the use of a mutated external tRNA, an external mutated RS, or a mutated external tRNA / RS pair. In such cases, the mutated external tRNA preferentially recognizes the degenerate codon for the wild-type tRNA, and introduces an unnatural amino acid into the protein in response to the degenerate codon, and wherein the external mutant synthetase preferentially aminoacylates the mutated external tRNA with an unnatural amino acid. In some embodiments, the unnatural amino acid is introduced into the polypeptide in vivo.

[0275] A wide selection of suitable reactive groups is known to those skilled in the art. Such suitable reactive groups may include, for example, amino, hydroxyl, carboxyl, carboxylate, carbonyl, alkenyl, alkynyl, aldehyde, ester, ether (e.g. thioether), amide, amine, nitrile, vinyl, sulfide, sulfonyl, phosphoryl, or the like chemically reactive groups. Additional suitable reactive groups include, but are not limited to, maleimide, N hydroxysuccinimide, sulfo-N-hydroxysuccinimide, nitrilotriacetic acid, activated hydroxyl, haloacetyl (e.g. bromoacetyl, iodoacetyl), activated carboxy, hydrazide, epoxide, aziridine, sulfonyl, trifluoromethyldiaziridine, pyridyldiulphide, N-acyl-imidazole, imidazolecarbamate, vinylsulfone, succinimidyl carbonate, isocyanate, azidium benzene , biotin and avidin.

[0276] In some embodiments, one of the reactive groups is an electrophilic moiety and the other reactive group is a nucleophilic moiety. The nucleophilic moiety or electrophilic moiety may be attached to the side chain of an unnatural amino acid; the corresponding group is then attached to the saccharide moiety.

[0277] Suitable electrophilic groups that react with nucleophilic groups to form a covalent bond are known to those skilled in the art. In certain embodiments, such electrophilic moieties include, but are not limited to, e.g., a carbonyl group, a sulfonyl group, an aldehyde group, a ketone group, a built-in ester group, a thioester group, a stable imino group, an epoxy group, an aziridine group, etc.

[0278] Suitable nucleophilic groups that can react with an electrophilic moiety are known to those of skill in the art. In certain embodiments, such nucleophiles include, for example, aliphatic or aromatic amines such as ethylenediamine. In certain embodiments, nucleophilic moieties include, but are not limited to, e.g. -NRI-NH2 (hydrazide), NR1 (C = O) NR2NH2 (semicarbazide), -NR1 (C = S) NR2NH2 (thiosemicarbazide), (C = O) NR1NH2 (carbonyl hydroxide d), - (C = S) NRINH2 (thiocarbonyl dragee d), (SO2) NR1NH2 (sulfonylhydrazide), -NR1 NR2 (C = O) NR3NH2 (carbazide), NR1NR2 (C = S) NR3NH2 (thiocarbazide), -O-NH2 (hydroxylamine), and the like, where R1, R2, and R3 are each independently H, or alkyl having 1-6 carbons, preferably H. In some embodiments, the reactive group is hydrazide, hydroxylamine, semicarbazide, carbohydrazide, sulfonylhydrazide or the like.

[0279] The reaction product between the nucleophilic and electrophilic moiety typically contains atoms originally present on the nucleophilic moiety. Typical bonds obtained by the reaction of aldehydes or ketones with nucleophilic moieties include reaction products such as oxime, amide, hydrazone, reduced hydrazone, carbohydrazone, thiocarbohydrazone, sufonylhydrazone, semicarbazone, thiosemicarbazone, or a similar functional group depending on the electrophilic grouping and the nucleophilic grouping used e.g. aldehyde, ketone, and / or the like) which is reacted with

-73 nucleophilic grouping. Bindings to carboxylic acids are usually referred to as carbohydrazides or as hydroxamic acids. Binding to sulfonic acids are usually called as sulfonylhydrazides or N-sulfonylhydroxylamines. The resulting binding can then be stabilized by chemical reduction.

[0280] These methods may further include contacting the saccharide moiety with a glycosyltransferase, saccharide donor moiety and other reagents required for glycosyltransferase activity for a sufficient period of time and under suitable conditions for transferring the saccharide from the saccharide donor moiety to the saccharide moiety. In certain embodiments, the method further comprises contacting the glycosyltransferase reaction product with at least a second glycosyltransferase and a second saccharide donor moiety. In other words, certain embodiments disclosed herein provide methods in which amino acids bound by saccharide moieties or an unnatural amino acid that contains a saccharide moiety is further glycosylated. These glycosylation steps are preferably (but not necessarily) carried out enzymatically using, for example, a glycosyltransferase, glycosidase, or other enzyme known to those skilled in the art. In some embodiments, multiple enzymatic steps are performed in one reaction mixture that contains two or more different glycosyltransferases. For example, a galactosylation and sialylation step can be performed simultaneously by including both sialyltransferase and galactosyltransferase in the reaction mixture.

[0282] For the synthesis of enzymatic saccharides that include a glycosyltransferase reaction, the recombinant cells optionally contain at least one heterologous gene that encodes the glycosyltransferase. Many glycosyltransferases are known as well as their polynucleotide sequences. See, e.g., "The WWW Guide To Cloned Glycosyl transferases," (available on the World Wide Web). The amino acid sequences of glycosyltransferases and the nucleotide sequences encoding glycosyltransferases from which the amino acid sequences can be derived are also listed in various publicly available databases, including GenBank, Swiss-Prot, EMBL, and others.

[0283] In certain embodiments, the glycosyltransferase includes, but is not limited to, e.g., galactosyltransferase, fucosyltransferase, glucosyltransferase, N-acetylgalactosaminyltransferase, N-acetylglucosaminyltransferase, glucuronyltransferase, glucuronyltransferase, Suitable glycosyltransferases include those derived from eukaryotes and prokaryotes.

[0284] Glucosyltransferase acceptors will be present on the glycoprotein to be modified by the methods disclosed herein. Suitable acceptors include, for example, galactosyl acceptors such as Gaipi, 4GalNAc-; Gal, 3GalNAc-; lacto-N-tetraoza-; Gal, 3GlcNAc-; Gal, 4GlcNAc-; Gal, 3Ara-; Gal, 6GlcNAc-; and Gaipi, 4Glc- (lactose). Other acceptors known to those of skill in the art (see, e.g., Paulson et al., J. Biol. Chem. 253:

-745617-5624, 1978). Typically, acceptors form part of the saccharide moiety chain that is attached to the glycoprotein.

[0285] In one embodiment, the saccharide moiety comprises a terminal GlcNAc, the saccharide donor moiety is UDP-GlcNAc and the glycosyltransferase is βΐ4N-acetylglucosaminyltransferase. In another embodiment, the saccharide moiety contains a GlcNAc terminal, the saccharide donor moiety is UDP-Gal and the glycosyltransferase is βΙ-4-galactosyltransferase. Additional sugars can also be added.

[0286] Glycosylation reactions include, in addition to the respective glycosyltransferase and acceptor, activated nucleotide sugar that acts as a sugar donor for the glycosyltransferase. The reactions may also contain other components that facilitate the activity of the proper glycosyltransferase. These components may contain a divalent cation (e.g., Mg²⁺ or Mn), materials needed for ATP regeneration, phosphate ions and organic solvents. The concentration or amount of the various reagents used in these processes depends on many factors, such as reaction conditions such as temperature and pH, and the selection and amount of glycosylation acceptor saccharides. The reaction medium may also contain solubilization detergents (e.g. Triton or SDS) and organic solvents such as methanol or ethanol, if necessary.

[0287] Also provided by certain embodiments for glycoprotein modification are compositions that contain a translation system that may or may not contain host cells, mutated external tRNA, external mutated RS or any or all of them.

[0288] As used herein, the term "saccharide moiety" refers to natural and unnatural saccharide moieties (i.e., an unnaturally occurring saccharide moiety, e.g. a saccharide moiety that is modified, e.g., at one or more hydroxyl positions or amine, e.g. dehydroxylated, deaminated, esterified, etc., e.g. 2-deoxyGal is an example of an unnatural saccharide moiety).

[0289] The term "carbohydrate" has the general formula (Cf + O), and includes, but is not limited to, e.g., monosaccharides, disaccharides, oligosaccharides and polysaccharides. Oligosaccharides have chains composed of sugar units that are alternatively referred to as sugars. Saccharide units can be arranged in any order and binding between two saccharide units can be carried out in any of about ten different ways. The following abbreviations are used here: Ara = arabinosyl; Fru = fructosyls; Fuc = fucosyl; Gal = galactosyl; GalNAc = N-acetylogalaktozaminyl; Glc = glucosyl; GlcNAc = N-acetylglucosaminyl; Man = mannozyl; and NeuAc = sialyl (typically N-acetone loneuramine 1).

[0290] Oligosaccharides are believed to have a reducing end and a non-reducing end, regardless of whether the saccharide at the reducing end is actually a reducing sugar. In accordance with the adopted nomenclature, the oligosaccharide structures are shown here graphically with a non-reducing end on the left and a reducing end on the right. All oligosaccharides described herein are described by the name or abbreviation for a non-reducing saccharide (e.g. Gal) followed by the configuration of the glycosidic bond (a or β), ring binding, ring position of the reducing saccharide involved in the binding, followed by the name or abbreviation of the reducing saccharide (e.g., GlcNAc). The bond between two sugars can be expressed, for example, as 2.3; 2 -&gt; - 3; 2-3; or (2,3). Natural and unnatural bonds (e.g., 1-2; 1-3; 1-4; 1-6; 2-3; 2-4; 2-6; etc.) between two sugars are included in some embodiments. Each of the saccharides is pyranose.

[0291] The term "sialic acid" (abbreviation "Sia") refers to each member of the family of carboxylated sugars with nine atoms. The most typical member of the sialic acid family is N-acetyl neuraminic acid (2-keto-5-acetamindo-3,5-dideoxy-D-glycero-D-galactononulopyranose-1-one) (often abbreviated as Neu5Ac, NeuAc or NANA). The second member of the family is N-glycolyl neuraminic acid (Neu5Gc or NeuGc), in which the NeuAc N-acetyl group is hydroxylated. The third member of the sialic acid family is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al., J. Biol. Chem. 261: 11550-11557, 1986; Kanamori et al., J. Biol. Chem. 265: 21811-21819, 1990). Also included are 9-substituted sialic acids such as 9-0C1-C6 acyl-Neu5Ac such as 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac, 9-deoxy-9-fluoroNeu5Ac and 9-azido-9 deoxy-Neu5Ac. For a review of the sialic acid family, see, e.g. Varki, Glycobiology 2: 25-40, 1992; Sialic Acids: Chemistry, Metabolism and Function, R. Schauer, Ed. (Springer-Verlag, New York (1992). The synthesis and use of sialic acid compounds in the sialing procedure is described in, for example, International Application WO 92/16640 (all contents incorporated herein by reference).

[0292] Donor substrates for glycosyltransferases are activated nucleotide sugars. Such activated sugars generally consist of uridine and guanosine diphosphate and cytidine monophosphate, sugar derivatives in which nucleoside diphosphate or monophosphate serves as a leaving group. Bacterial, plant or fungal systems can sometimes use other activated nucleotide sugars.

[0293] The introduction of an unnatural amino acid, e.g. an unnatural amino acid containing a moiety, wherein the saccharide moiety can be attached, or an unnatural amino acid that contains a saccharide moiety, can be performed to, e.g., adjust changes in the structure and / or function of the protein, e.g. to change size, acidity, nucleophilicity, hydrogen bonding, hydrophobicity, availability of protease targets, target access to the protein moiety, etc. Proteins that contain an unnatural amino acid, e.g., an unnatural amino acid containing a moiety, wherein the saccharide moiety can be attached, or an unnatural amino acid that contains a saccharide moiety, may have enhanced, or even completely new, catalytic or physical properties.

[0294] For example, the following properties are optionally modified by the inclusion of an unnatural amino acid, e.g., an unnatural amino acid containing a moiety, wherein the saccharide moiety can be attached, or an unnatural amino acid that

- contains a saccharide moiety, to protein: toxicity, biodistribution, structural properties, spectroscopic properties, chemical and / or photochemical properties, catalytic ability, half-life (e.g. serum half-life), ability to react with other molecules, e.g. covalently or non-covalent and similar. Compositions containing proteins that contain at least one unnatural amino acid, e.g. an unnatural amino acid containing a moiety, wherein the saccharide moiety can be attached, or an unnatural amino acid that contains a saccharide moiety, are useful for, e.g., new therapeutic agents, diagnostic agents, catalytic enzymes, industrial enzymes, binding proteins (e.g., antibodies), and protein structure and function studies. See, e.g., Dougherty, Curr. Opin. in Chem. Biol., 4: 645-652 (2000).

[0295] In one aspect, the composition comprises at least one protein with at least one, e.g., at least about two, three, four, five, six, seven, eight, nine, or at least about ten or more unnatural amino acids, for example. unnatural amino acids containing a moiety, wherein the saccharide moiety can be attached, or unnatural amino acids that contain a saccharide moiety, and / or that contain another unnatural amino acid. Unnatural amino acids can be the same or different, e.g. there can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more different sites in a protein containing 1, 2, 3, 4, 5, 6 , 7, 8, 9, or 10 or more different unnatural amino acids. In another aspect, the composition comprises a protein with at least one, but less than all, of specific amino acids in a protein substituted with an unnatural amino acid, e.g. For a given protein with more than one unnatural amino acid, the unnatural amino acids may be the same or different (e.g. the protein may contain two or more different types of unnatural amino acids, or it may contain two of the same unnatural amino acids). For a given protein with two or more unnatural amino acids, the unnatural amino acids may be the same, different, or may be a combination of multiple unnatural amino acids of the same type with at least one different unnatural amino acid.

[0296] Substantially any protein (or portion thereof) that contains an unnatural amino acid, e.g., an unnatural amino acid containing a moiety, wherein the saccharide moiety is attached, such as an aldehyde or ketone derivatized amino acid, or an unnatural amino acid that contains a saccharide moiety (and any corresponding coding for nucleic acid, e.g. which contains one or more selector codons) can be produced using the compositions and methods described herein. No attempt is made to identify hundreds of thousands of known proteins, each of which can be modified to contain one or more unnatural amino acids, e.g. by adapting all available mutation methods to contain one or more of the appropriate degenerate codons in the appropriate translation system. Typical sequence repositories for known proteins include GenBank EMBL, DDBJ and NCBI. Other repositories can be easily identified by searching the internet.

[0297] Typically, the proteins are, e.g., at least about 60%, 70%, 75%, 80%, 90%, 95%, or at least about 99% or more identical to any available protein (e.g. therapeutic protein, diagnostic protein, industrial enzyme or fragment thereof, and the like) and contain one or more unnatural amino acids.

[0298] In addition to modifying one or more amino acid residues of a protein, the carbohydrate composition of the protein may be modified, ie, by glycosylation. Post-translational modifications of proteins by glycosylation methods can affect protein folding and stability, modification of protein internal activity and modulation of their interaction with other biomolecules. See, e.g., Varki, Glycobiology 3: 97-130, 1993, hereby incorporated by reference in its entirety. Natural glycoproteins are often present in a population of different glycoforms, which makes glycan structure analysis and glycosylation effects on protein structure and function difficult. Thus, methods for the synthesis of natural and unnatural homogeneously glycosylated proteins are needed to systematically understand the function of glycans, and to develop improved therapeutic agents for glycoproteins.

[0299] One class of proteins that can be made using certain compositions and methods disclosed herein includes transcription modulators, enzymes, or a portion thereof. Examples of transcription modulators include genes and proteins of transcription modulators that modulate cell growth, differentiation, regulation, or the like. Transcription modulators occur in prokaryotes and viruses and eukaryotes, including fungi, plants, yeasts, insects and animals, including mammals, providing a range of therapeutic goals. It should be noted that expression and transcriptional activators regulate the transcription of many mechanisms, e.g. by binding to receptors, stimulating the signal transduction cascade, regulating expression of transcription factors, binding of promoters and enhancers, binding to proteins that bind to promoters and enhancers, DNA inversion, pre-mRNA splicing, RNA polyadenylation and RNA degradation. Some examples of enzymes include, but are not limited to, e.g. Amidases, amino acid racemases, acylases, dehalogensases, dioxygenases, diaryl propane peroxidases, epimerases, epoxy hydrolases, esterases, isomerases, kinases, glucose isomerases, glycosidases, glycosyltransferases, haloperoxidase, monooxygenase, and nitoxygenase, e.g. , proteases, phosphatases, subtilisins, transaminases and nucleases.

[0300] Some of these polypeptides, which may be modified depending on certain embodiments disclosed herein, are commercially available (see, e.g., Sigma Biosciences catalog and price list), and the respective protein sequences and genes, and usually many variations thereof , is well known (see, e.g., Genbank).

[0301] Examples of therapeutically relevant properties that can be modified by manipulation or any embodiment disclosed herein (including glycosylation and / or pegylation, and / or the introduction of unnatural amino acids) include serum half-life, storage half-life, stability, immunogenicity , therapeutic activity, detectability (e.g., by grouping

Reporters (e.g., tags or tag binding sites) in unnatural amino acids, specificity, reduced LD50 or other side effects, ability to enter the body through the gastrointestinal tract (e.g., oral availability), or the like. Examples of suitable diagnostic properties include storage half life, stability, diagnostic activity, detectability, specificity or the like. Examples of suitable enzymatic properties include storage half-life, stability, specificity, enzymatic activity, production capacity or the like.

[0302] Many other proteins can also be modified to contain one or more unnatural amino acids according to certain embodiments disclosed herein. For example, proteins from infectious fungi, e.g., Aspergillus, Candida species, bacteria, especially E. coli, which serve as a model for pathogenic bacteria as well as medically important bacteria such as Staphylococci (e.g. aureus) or Streptococci (e.g. pneumoniae); protozoa such as sporozoa (e.g. Plasmodia), rootlets (e.g. Entamoeba) and flagellates (Trypanosoma, Leishmania, Trichomonas, Giardia, etc.); viruses such as (+) RNA viruses (examples include poxviruses, e.g., vaccinia; Picomaviruses, e.g. polio; Togaviruses, e.g. rubella; Flaviviruses, e.g. HCV; and Coronaviruses), RNA viruses (e.g. Rabdoviruses, e.g. VSV; Paramyxoviruses, e.g. RSV; Ortomixoviruses, e.g. influenza; Buniaviruses; and Arenaviruses), dsDNA viruses (Reoviruses, e.g.), RNA viruses for DNA, i.e. Retroviruses, e.g. HIV and HTFV, and certain DNA to RNA viruses such as hepatitis B virus.

[0303] Agricultural-related proteins such as insect resistance proteins (e.g. Cry proteins), enzymes for starch and lipid production, plant and insect toxins, toxin resistance proteins, mycotoxin detoxification proteins, plant growth enzymes (e.g. ribulose 1, Carboxylase / oxygenase, "RUBISCO"), lipoxygenase (LOX), and phosphoenolpyruvate (PEP) carboxylase are also suitable modification targets by some of the embodiments disclosed herein.

[0304] In certain embodiments, the protein or polypeptide of interest (or portion thereof) in the methods and / or compositions disclosed herein is encoded by a nucleic acid. Typically, the nucleic acid contains at least one degenerate codon, at least about two, three, four, five, six, seven, eight, nine or at least about ten or more degenerate codons.

[0305] Thus, the above-described artificial (e.g., human-made, non-naturally occurring) polypeptides and polynucleotides are also features of some of the embodiments disclosed herein. An artificial polynucleotide may include, e.g. (a) a polynucleotide comprising a nucleotide sequence encoding an artificial polypeptide; (b) a polynucleotide that is complementary to or which encodes a polynucleotide sequence (a); (c) a nucleic acid that hybridizes to the polynucleotide (a) or (b) under conditions of contact that substantially over the entire length of the nucleic acid; (d) a polynucleotide that is at least about 95%, preferably at least about 98% identical to polynucleotide (a), (b) or (c); and, (e) a polynucleotide comprising a conservative change (a), (b), (c) or (d).

[0306] Unnatural amino acids are generally described above. Particularly interesting for glycoprotein production, as described herein, are unnatural amino acids in which R in Formula I contains a group that can react with a reactive group attached to a saccharide moiety to bind the saccharide moiety to a protein that contains an unnatural amino acid. Suitable R groups include, for example, keto-, azido, -hydroxyl, hydrazine, cyano, halo-, aminooxy, alkenyl, alkynyl, carbonyl, ether, thiol, selenium, sulfonyl, borate, boronate, phospho, phosphono , phosphine, heterocycle, enone, imine, aldehyde, ester, thioacid, thioester, built-in ester, hydroxylamine, amine, and the like, or any combination thereof. In some embodiments, the unnatural amino acids comprise a photoactivated cross-linking agent.

[0307] In addition to unnatural amino acids that contain new side chains, unnatural amino acids also optionally contain modified skeletal structures, e.g. as represented by structures of Formula II and III:

<img file="PL1999259T3_D0003.tif" />

WzórD

Formulain where Z is typically OH, NH2, SH, NH-R 'or S-R'; X and Y, which may be the same or different, contain S or O, and R and R ', which are optionally the same or different, are usually selected from the same list of substituents for the R group described above for the unnatural amino acids of Formula I and hydrogen. For example, the unnatural amino acids disclosed herein optionally have amino or carboxy substitutions as shown by Formulas II and III. Unnatural amino acids of this type include, but are not limited to, α-hydroxy acids, α-thio acids, and aminothiocarboxylates, e.g., with side chains corresponding to the side chains of twenty typical natural or unnatural amino acids. In addition, substitutions at α-carbon optionally include L, D, or α-α-disubstituted amino acids such as D-glutamate, D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and the like. Other structural alternatives include cyclic amino acids such as proline analogs and proline analogues with a 3, 4, 6, 7, 8 and 9-membered ring, β and γ amino acids such as substituted β-alanine and γ-aminobutyric acid.

[0308] For example, many unnatural amino acids are based on natural amino acids such as tyrosine, glutamine, phenylalanine and the like. Tyrosine analogs include para-substituted tyrosines, ortho-substituted tyrosines, and meta-substituted tyrosines, wherein the substituted tyrosine contains an acetyl group, benzoyl group, amino group, hydrazine, hydroxyamine, thiol group, carboxyl group, isopropyl group, methyl group, C6- C20 straight chain or branched hydrocarbon,

Unsaturated or unsaturated hydrocarbon, O-methyl group, polyether group, nitro group, or the like. In addition, multi-substituted aryl rings are also contemplated. Glutamine analogs include, but are not limited to, α-hydroxy derivatives, γ-substituted derivatives, cyclic amide-substituted derivatives and glutamine derivatives. Exemplary phenylalanine analogs include, but are not limited to, meta-substituted, ortho-substituted and / or para-substituted phenylalanines, the substituent having a hydroxyl group, methoxy group, methyl group, allyl group, aldehyde or ketone group or the like.

[0309] Specific examples of unnatural amino acids include, but are not limited to, p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3- (2-naphthyl) alanine, 3-methyl-phenylalanine, Ο-4-allyl -L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcPserine, β-Ο-GlcNAc-L-serine, tri-O-acetyl-GalNAc-a-threonine, and-GalNAc-L-threonine , LDopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, pacyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, listed below or elsewhere in this text and the like.

[0310] Unnatural amino acids suitable for use in some of the methods disclosed herein also include those having a saccharide moiety attached to the amino acid side chain. In one embodiment, the unnatural amino acid with a saccharide moiety comprises the amino acid serine or threonine, Man, GalNAc, Gic, Fuc or Gal. Examples of unnatural amino acids that contain a saccharide moiety include, but are not limited to, e.g. tri-O-acetylΰΙοΝΑοβ-βε ^^, β-Ο-GlcNAc-L-serine, tri-O-acetyl-GalNAc-α-threonine, a-GalNAc-Ltreonine, O-Man-L-serine, tetra-acetyl- O-Man-L-serine, O-GalNAc-L-serine, tri-acetylO-GalNAc-L-serine, Glc-L-serine, tetraacetyl-Glc-L-serine, fuc-L-serine, tri-acetyl fucL-serine, O-Gal-L-serine, tetra-acetyl-O-Gal-L-serine, beta-O-GlcNAc-L-threonine, triacetyl-beta-GlcNAc-L-threonine, O-Man-L- threonine, tetra-acetyl-O-Man-L-threonine, OGalNAc-L-threonine, tri-acetyl-O-GalNAc-L-threonine, Glc-L-threonine, tetraacetyl-Glc-Ltreonine, fuc-L-threonine, tri-acetyl-fuc-L-threonine, O-Gal-L-threonine, tetra-acetyl-O-Gal-Lserine and the like. Some embodiments also include unprotected and acetylated forms of the above.

[0311] In some embodiments, the design of unnatural amino acids is directed by known information about active synthetase sites, e.g., external mutant tRNA synthetases used to aminoacylate the mutated external tRNA. For example, three classes of glutamine analogues are provided, including derivatives substituted on the amide nitrogen atom (1), the methyl group in the position γ (2) position, and aN-Cy-cyclic derivative (3). Based on the X-ray crystal structure of GlnRS E. coli, in which the main residue binding site is homologous to yeast GlnRS, analogs are designed to complement a number of residue side chain mutations within the 10 A glutamine side chain coating, e.g., mutation of the Phe233 active site to small

The 81-hydrophobic amino acid may be supplemented by an increase in the spatial volume at the Cy position for Gin.

[0312] For example, 1,5-N-phytoyl-L-glutamic anhydride (compound number 4 in FIG. 23 of WO 02/085923) is optionally used for the synthesis of glutamine analogues with substituents on the amide nitrogen. See, e.g., King and Kidd, J. Chem. Soc., 3315-3319, 1949; Friedman and Chatterrji, J. Am. Chem. Soc. 81, 3750-3752, 1959; Craig et al., J. Org. Chem. 53, 1167-1170, 1988; and Azoulay et al., Eur. J. Med. Chem. 26, 201-5, 1991. The anhydride is usually prepared from glutamic acid by first protecting the amine as a phthalimide and then by heating to reflux in acetic acid. The anhydride then opens with many amines to give a number of different substituents on the amide. By deprotecting the phalooyl group using hydrazine, a free amino acid is obtained, as shown in FIG. 23 of WO 2002/085923.

[0313] The substitution at the γ position is usually carried out by alkylation of glutamic acid. See, e.g., Koskinen and Rapoport, J. Org. Chem. 54, 1859-1866, 1989. A protected amino acid, e.g., as shown in compound 5 in FIG. 24 of WO 02/085923, optionally obtained first by alkylation of the amino moiety with 9-bromo-9-phenylfluorene (PhflBr) (see, e.g., Christie and Rapoport, J. Org. Chem. 1989, 1859-1866, 1985), and then esterifies the acid groups using O-tert-butyl-Ν, diisopropylisourea. Addition of KN (Si (CH3) 3)₂ regioselectively deprotonates in the β position of the methyl ester to form an enolate, which is then optionally alkylated with a series of alkyl iodides. Hydrolysis of the t-butyl ester and the Phfl group gives the desired? -Methyl glutamine analogue (Compound No. 2 in FIG. 24 of WO 02/085923).

[0314] Certain other embodiments include an immunoconjugate comprising an antibody (or functional fragment thereof) specific for the target (e.g., target cell), the antibody (or fragment thereof or functional equivalent thereof) conjugated, in specific, predetermined positions, two or more the number of therapeutic molecules, each of which contains an unnatural amino acid. In certain embodiments, the antibody fragments are F (ab ') 2, Fab', Fab or Fv fragments.

[0315] In certain embodiments, two or more therapeutic molecules are the same. In certain embodiments, two or more therapeutic molecules are different. In certain embodiments, therapeutic molecules are conjugated to the same unnatural amino acid. In certain embodiments, therapeutic molecules are conjugated to various unnatural amino acids.

[0316] In some embodiments, the nature or binding chemistry of the unnatural amino acid / therapeutic molecule allows cleavage of the binding under certain conditions, such as mild or weak acid conditions (e.g., about pH 4-6, preferably about pH5), a reducing environment (e.g. presence of reducing agent) or divalent cations and is accelerated by heat, optionally.

[0317] In certain embodiments, the therapeutic molecule is conjugated to the antibody via a linker / spacer (e.g., one or more methylene repeats (CH₂-), methyleneoxy (-CH2-O-), methylene carbonyl (-CH2-CO-), amino acids or combinations thereof).

Multi-protein complexes [0318] Unnatural amino acids can also be used to combine two or more proteins or protein subunits with unique functions. For example, bispecific antibodies can be produced by combining two antibodies (or functional parts thereof, or derivatives thereof, such as Fab, Fab ', Fd, Fv, scFv fragments, etc.) by introduced unnatural amino acids.

[0319] Thus, certain embodiments herein provide methods for synthesizing multiple protein conjugates. These methods use, in some embodiments, the introduction into a first protein (e.g., a first antibody) of a first unnatural amino acid that contains a first reactive group; and contacting the first protein with the second protein (e.g. second antibody) containing a second unnatural amino acid that contains a second reactive group, the first reactive group reacting with the second reactive group, thereby forming a covalent bond that attaches the second protein to the first protein.

[0320] The first reactive group is, in some embodiments, an electrophilic moiety (e.g., a ketone moiety, aldehyde moiety, and / or the like) and the second reactive group is a nucleophilic moiety. In some embodiments, the first reactive group is a nucleophilic moiety and the second reactive group is an electrophilic moiety (e.g., ketone moiety, aldehyde moiety and / or the like). For example, the electrophilic moiety is attached to the unnatural amino acid of the first Ab, and the nucleophilic moiety is attached to the unnatural amino acid of the second Ab.

[0321] Different functional domains from different proteins can be linked together by a similar method to create new proteins with new functions (e.g., new transcription factors with a unique combination of binding domains and activation of DNA transcription; new enzymes with new regulatory domains, etc.).

PH Sensitive Binding [0322] Many protein interactions are pH sensitive, in the sense that the binding affinity of one protein for its usual binding partner may vary depending on changes in the pH of the environment. For example, many ligands (such as insulin, interferons, growth hormone, etc.) bind their respective receptors on the cell surface to induce signal transduction. The ligand-receptor complex is then internalized by receptor-mediated endocytosis and passes through a series of increasingly acid endosomes. Finally, the ligand-receptor interaction is attenuated at a specific acidic pH (e.g., around pH 5.0) and the ligand dissociates from the receptor. Some receptors (and perhaps some Ugandans) may be recycled to

-83 cell surface. There they may be able to bind their respective normal binding partners.

[0323] If the pH-sensitive binding can be modulated such that the receptor ligand can be dissociated at a relatively higher pH, then some ligands may dissociate earlier from their receptors and preferably return to the cell surface rather than degrade. This will lead to an increased in vivo half-life of such ligands, which may be desirable as less insulin may be needed for the same (or better) efficacy in diabetic patients. In other cases, it may be desirable to modulate pH sensitive binding by promoting lower pH binding.

[0324] For example, monoclonal antibodies are generally very specific for their purposes. However, in many applications, for example in cancer therapy, they tend to cause some side effects by, for example, binding to non-cancerous tissues. One reason may be that the tumor targets against which monoclonal antibodies are generated are not specifically expressed in cancer cells, but also expressed (although there may be fewer) in certain healthy cells. Such side effects are usually undesirable and there is a need for antibodies with better specificity.

[0325] The pH of human blood is tightly regulated and maintained at around 7.6-7.8. On the other hand, tumor cells exhibit extracellular pH 6.3-6.5 due to the accumulation of metabolic acids, which are inefficient due to poor tumor vasculature. If the interaction between the tumor antigen and its therapeutic antibody can be modulated such that binding is preferred at low pH, the tumor-antibody may have additional specificity / affinity / selectivity for these tumor antigens, although the same tumor antigens sometimes also occur in normal tissues.

[0326] In fact, such modified antibodies may be desirable not only for cancer therapy, but also desirable for antigen-antibody binding that can occur at a lower than normal pH level.

General techniques [0327] Practice of the embodiments disclosed herein will utilize, unless otherwise indicated, conventional techniques of molecular biology, cell biology, cell cultures, microbiology and recombinant DNA, which are within the skill of one of ordinary skill in the art. Such techniques are fully explained in the literature. See, for example, Molecular Cloning: A Laboratory Manual, 2nd Ed., Ed. By Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (DN Glover ed., 1985); Oligonucleotide Synthesis (MJ Gait ed., 1984); Mullis et al .; U.S. Patent No.: 4,683,195; Nucleic Acid Hybridization (BD Hames &amp; SJ Higgins eds. 1984); Transcription And Translation (BD Hames &amp; SJ Higgins eds. 1984); B. Perbal, A Practical Guide To Molecular Cloning (1984); dissertation, Methods In Enzymology (Academic Press, Inc., NY); Methods In Enzymology, Vol. 154 and 155 (Wu et al., Eds.),

-84 Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987).

[0328] In addition, general texts revealing general cloning, mutation, cell culture and the like include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology vol. 152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning-A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 2000 ("Sambrook") and Current Protocols in MolecularBiology, FM Ausubel et al., Eds., Current Protocols, joint venture Greene Publishing Associates, Inc. and John Wiley &amp; Sons, Inc., (supplemented to 2002) ("Ausubel")) are all incorporated herein by reference in their entirety. These texts describe mutagenesis, the use of vectors, promoters and many other important issues related to, e.g., generation of mutated external tRNA, external mutated synthetase and their pairs.

[0329] Various types of mutagenesis are used in certain embodiments, e.g., to create new synthetases or tRNAs. These include, but are not limited to, site-directed (such as the use of an Amber, Ochra, Umber or other stop codon), wobble codon mutagenesis, random point mutagenesis, homologous recombination (DNA shuffling), mutagenesis using uracil-containing matrices, oligonucleotide targeted mutagenesis. phosphorothioate-modified DNA mutagenesis, gapped duplex DNA or similar. Additional suitable methods include mismatch point repair, mutagenesis using repair deficient host strains, limiting-selection and limiting purification, deletion mutagenesis, total gene synthesis mutagenesis, double strand break repair and the like. Mutagenesis, e.g. involving chimeric constructs, is also included in some embodiments. In one embodiment, mutagenesis can be directed using known information of a naturally occurring molecule or altered or mutated naturally occurring molecule, e.g., sequence, sequence comparisons, physical properties, crystal structure or the like.

[0330] The above texts and examples describe these procedures as well as the following publications and the literature cited therein: Sieber, et al., Nature Biotechnology, 19: 456-460 (2001); Ling et al., Approaches to DNA mutagenesis: an overview, Anal. Biochem. 254 (2): 157-178 (1997); Data et al., Methods Mol. Biol. 57: 369-374 (1996); IA Lorimer, I. Pastan, Nucleic Acids Res. 23, 3067-8 (1995); WPC Stemmer, Nature 370, 389-91 (1994); Arnold, Curr. Opin. in Biotech. 4: 450-455 (1993); Bass et al., Science 242: 240-245 (1988); Fritz et al., Nuci. Acids Res. 16: 6987-6999 (1988); Kramer et al., Nuci. Acids Res. 16: 7207 (1988); Sakamar and Khorana, Nuci. Acids Res. 14: 6361-6372 (1988); Sayers et al., Nuci. Acids Res. 16: 791-802 (1988); Sayers et al., Nuci. Acids Res. 16: 803-814 (1988); Carter, Methods in Enzymol. 154: 382-403 (1987); Kramer &amp; Fritz Methods in Enzymol. 154: 350-367 (1987); Kunkel, Nucleic Acids &amp; Mol. Biol. (Eckstein, F. and Lilley, DMJ eds., Springer Verlag, Berlin)) (1987); Kunkel et al., Methods in Enzymol. 154, 367-382 (1987); Zoller &amp; Smith, Methods in Enzymol. 154: 329-350 (1987); Carter

-85Biochem. J. 237: 1-7 (1986); Eghtedarzadeh &amp; Henikoff, Nuci. Acids Res. 14: 5115 (1986); Mandecki, PNAS, USA, 83: 7177-7181 (1986); Nakamaye &amp; Eckstein, Nuci. Acids Res. 14: 9679-9698 (1986); Wells et al., Phil. Trance. R. Soc. Lond. A 317: 415-423 (1986); Botstein &amp; Shortle, Science 229: 1193-1201 (1985); Carter et al., Nuci. Acids Res. 13: 4431-4443 (1985); Grundstróm et al., Nuci. Acids Res. 13: 3305-3316 (1985); Kunkel, PNAS, USA 82: 488-492 (1985); Smith, Ann. Rev. Genet. 19: 423-462 (1985); Taylor et al., Nuci. Acids Res. 13: 8749-8764 (1985); Taylor et al., Nuci. Acids Res. 13: 8765-8787 (1985); Wells et al., Gene 34: 315-323 (1985); Kramer et al., Nuci. Acids Res. 12: 9441-9456 (1984); Kramer et al., Cell 38: 879-887 (1984); Nambiar et al., Science 223: 1299-1301 (1984); Zoller &amp; Smith, Methods in Enzymol. 100: 468-500 (1983); and Zoller &amp; Smith, Nuci Acids Res. 10: 6487-6500 (1982). Additional information on many of the above methods can be found in Methods in Enzymology Volume 154, which also describes useful troubleshooting controls for various mutagenesis methods.

[0331] Oligonucleotides, e.g. for use in mutagenesis in certain embodiments, e.g., mutation of a synthetase library or tRNA alteration, are usually chemically synthesized according to the solid phase method of the phosphoramidate phosphate triester described by Beaucage and Caruthers, Tetrahedron Letts. 22 (20): 1859-1862, (1981) e.g. using an automated synthesizer as described in Needham-VanDevanter et al., Nuci Acids Res., 12: 6159-6168 (1984).

[0332] Furthermore, in principle any nucleic acid can be any or standard ordered from any of a variety of commercial sources such as The Midland Certified Reagent Company, The Great American Gene Company, ExpressGen Inc., Operon Technologies Inc. (Alameda, Calif.) And many others.

[0333] All of the embodiments described herein are intended to be combined with one or more other embodiments, even for those described in various parts of this disclosure.

[0334] All of the above US patents, US patent application publications, US patent applications, foreign patents, foreign patent applications and non-patent publications referred to herein and / or listed in the Application Data Sheet are incorporated herein by reference in their entirety . EXAMPLES [0335] These examples illustrate the introduction of an amino acid analog into proteins at codon-encoded positions that normally specifically encode phenylalanine (Phe) or specifically encode tryptophan (Trp). The scheme is shown in Figure 1. A similar approach can be used for other analogues.

[0336] Phe is encoded by two codons, UUC and UUU. Both codons are read by a single tRNA which is equipped with a GAA anti-codon sequence. The UUC codon is therefore recognized by standard Watson-Crick pairing between the codon and the anti-codon; UUU is read by a fluctuating pair of GU bases in the first position of anti-codone (Crick, J. Mol. Biol. 19: 548, 1966; Soli and RajBhandary, J. Mol.

-86Biol. 29: 113, 1967). Thermal denaturation of RNA duplexes gave Gibbs free energy estimates for melting GU, GC, AU, and AC base pairs as 4.1, 6.5, 6.3 and 2.6 kcal / mol, respectively, at 37 ° C. Thus, the hesitating base pair, GU, is less stable than the Watson-Crick, AU base pair. Modified tRNA^phe equipped with AAA anti-codon (tRNA^pheAAA) was designed by genetic engineering to read the UUU codon and was designed to read these codons faster than the wild-type tRNA^pheGAA · [0337] Mouse dihydrofolate reductase (mDHFR), which contains nine Phe residues, was selected as the test protein. The expression of plasmid pQE16 encodes mDHFR under the control of the T5 bacteriophage promoter; this protein is equipped with the C-terminal hexahistidine (HIS₆) to facilitate purification by immobilized metal affinity chromatography.

[0338] Modified yeast PheRS (mu-yPheRS) was prepared by introducing the Thr415Gly or Thr415Ala mutation into the α subunit of the synthetase (Datta et al., J. Am. Chem. Soc. 124: 5652, 2002). The kinetics of Nal and Phe activation by mu-yPheRS were analyzed in vitro using an adenosine triphosphate-pyrophosphate exchange assay. It was found that the specificity constant (k_cat! K \, i) for Nal activation by mu-yPheRS is 1.55 χ 10 '(s' (M'), 8 times greater than that for Phe. Therefore, when the Nal to Phe ratio in the culture medium is high, ytRNA^pheAAA should be loaded mainly Nal. In addition, the T415G mutant was generated by mutagenesis of four primers.

[0339] Both E. coli and yeast synthetase are 0 ^ 2 hetero-tetramers and the molecular weights of each subunit are different a (ePheRS) = 37 kDA; a (yPheRS) = 57 kDa; β (ePheRS) = 87 kDa; and β (yPheRS) = 67.5, all approximately.

[0340] Accordingly, the following examples are provided by way of illustration and are not limiting.

EXAMPLE 1 [0341] To change the ability of the yeast aminoacyl tRNA synthetase, the yPheRS gene was amplified from the pUC-ASab2 matrix plasmid encoding the PheRS gene alpha and beta subunits. Amplification was carried out with an intergenic 14 base pair sequence containing a translation reinitiation site upstream of the ATG start code in the beta subunit gene.

[0342] The following oligo primers were used for PCR: 5'-CGA TTT TCA CAC AGG ATC CAG ACC ATG ATT CTA G-3 '(SEQ ID NO: 7) (starter 1 with BamEE restriction site) and 5'GAC GGC CAG TGA ATT CGA GCT CGG TAC-3 '(SEQ ID NO: 8) (primer 2 with AywI restriction site). The resulting DNA product was introduced into the BamEE and Kpnl pQE32 sites giving pQE32-yFRS. The mutated yPheRS polynucleotide was generated using the mutagenesis primer using standard techniques.

[0343] Briefly, two complementary oligonucleotides: 5'-CTA CCT ACA ATC CTT ACG GCG AGC CAT CAA TGG AAA TC-3 '(SEQ ID NO: 9) (starter 3) and 5'-GAT TTC CAT TGA TGG CTC GCC GTA AGG ATT GTA GGT AG-3 '(SEQ ID NO: 10) (starter 4)

Synthesized to transfer a specific mutation at position 415 of the alpha subunit of the yPheRS polynucleotide.

EXAMPLE 2 [0344] Each plasmid pQE32-yFRS, and pQE32-T415G, pQE32-T415A was transformed into an E. coli BLR host cell (from NOVAGEN®) to create BLR expression strains (pQE32-yFRS_i BLR (pQE32-T415G) Cells. cultured in LB medium to a concentration of 0.6 at OD 600. Expression was then induced with 1 mM IPTG for 4 hours. Cells were collected and the polypeptides were purified using a nickel-nitrilotriacetic acid affinity column under native conditions according to the manufacturer's protocol (QIAGEN®). Imidazole in elution buffer was removed by desalting column, and the polypeptides were eluted in buffer containing 50 mM Tris-HCl (pH = 7.5), 1 mM DTT. Polypeptide samples were stored at -80 ° C with 50% glycerol.

EXAMPLE 3 [0345] The amino acid-dependent ATP-PPj exchange reaction was used to evaluate the activation of unnatural amino acids by yPheRS. The test was carried out in 200 microliters of reaction buffer containing 50 mM HEPES ((pH = 7.6), 20 mM MgCf, 1 mM DTT, 2 mM ATP and 2 mM [³²P] -PPj with specific activity 0.2-0.5 TBq / mol. Depending on the activity of various unnatural amino acids with synthetase, the amino acid concentration ranged from 10 microM to 5 mM and the enzyme concentration ranged from 10 nM to 100 nM. 20 microliter samples were removed from the reaction solution at various time points and the reaction was terminated by the addition of 500 microliters buffer solution containing 200 mM NaPPj, 7% w / v HClO4 and 3% w / v activated carbon. The carbon was centrifuged and washed twice with 500 microliters of 10 mM NaPPj and 0.5% HClO4 solution. Radiolabeled ATP absorbed by carbon was quantified by liquid scintillation methods. Specificity constants were calculated by fitting nonlinear data regression to the Michaelis Menten model. The kinetic parameters for the exchange of ATP-PPi and amino acids using yPheRS (T415G), wild type yPheRS and the yPheRSnaph variant are shown in the table below.

<td>Amino acid</td><td>Enzyme</td><td>Km (μΜ)</td><td>Kcat (s'¹)</td><td>Kcat / Km (M '* s'')</td><td>Kcat / Km (relative</td>

<td>phe</td><td>T415G</td><td> 55+/-14</td><td> 0.202+/-0.11</td><td> 3512+/-1134</td><td>l^and</td>

<td>Trp</td><td>T415G</td><td> 2.83+/-1.6</td><td> 0.153+/-0.003</td><td> 63190+/-34590</td><td> 18^and</td>

<td>2Nal</td><td>T415G</td><td> 7.03+/-0.14</td><td> 0.208+/-0.04</td><td> 29535+/-5848</td><td> 8.4^and</td>

<td>phe</td><td>wild type</td><td> 3.85+/-0.99</td><td> 0.181+/-0.011</td><td> 50994+/-22655</td><td> 15^and</td>

<td>phe</td><td>Naph</td><td> 11010+/2688</td><td> 0.0095+/0.0021</td><td> 0.855+/-0.007</td><td>l^b</td>

<td>Trp</td><td>Naph</td><td> 1424+/-597 .</td><td> 0.0035+/-</td><td> 2.52+/-0.44</td><td> 2.9^b</td>

<td>Amino acid</td><td>Enzyme</td><td>Km (pM)</td><td>Kcat (s'¹)</td><td>Kcat / Km (M''s'</td><td>Kcat / Km (relative</td>

<td></td><td></td><td></td><td> 0.0009</td><td></td><td></td>

<td>2Nal</td><td>Naph</td><td> 2030+/-691</td><td> 0.030+/-0.018</td><td> 14.54+/-4.22</td><td> 17^b</td>

EXAMPLE 4 [0346] The expression plasmid, pQE16 (QIAGEN®) was used with the murine dihydrofolate reductase (mDHFR) polypeptide marker with the C-terminal hexa-histidine tag gene under the control of the T5 bacteriophage promoter and the terminator.

[0347] The Amber codon (TAG) was placed at position 38. mDHFR using the QUICK-CHANGE® mutagenesis kit. Two complementary oligo primers (5'-CCG CTC AGG AAC GAG TAG AAG TAC TTC CAA AGA ATG-3 '(SEQ ID NO: 11) and 5'-CAT TCT TTG GAA GTA CTT CTA CTC GTT CCT GAG CGG-3' ( SEQ ID NO: 12)) was used to prepare pQE16am. The mutated yPheRS T415G gene was amplified from pQE32-T415G and the constitutive tac promoter with the lac repressor binding site removed was added upstream of the gene start codon.

[0348] The entire T415G expression cassette was inserted into the Pvul \ pQE16 site giving pQE16amT415G. Mutant yeast suppressor tRNA (wwtRNA^phe(CUA)) was constitutively expressed under the control of the Ipp promoter. Expression cassette in RNA^phe(CUA) was inserted into the repressor plasmid pREP4 to form pREP4-tRNA using known methods.

[0349] Phenylalanine (Phe) auxotrophic bacterial strain; AF (KIO, Hfr (Cavalli) pheS93rel-1 tonA22 thi T2^R pheAlS) was used as the host strain. Double auxotrophic Phe / Trp strain, AFW (KIO, Hfr (Cavalli) pheS13rel-i tonA22 thi T2^R/? AeA18, trpBWA) and triple auxotrophic strain Phe / Trp / Lys AFWK (KIO, Hfr (Cavalli) pheS13rel- \ tonA22 thi T2^R phe AIS, trpB114, lysAj was obtained by P1-mediated transduction with trpB :: TnlO and lysA :: TnlO transposons.

EXAMPLE 5 [0350] Auxotrophic host cell strains AF, AFW and AFWK, each were transformed with the plasmid pQE16am-T415G and pREP4-tRNA to give the AF expression strains [pQE16am-T415G / pREP4-tRNA] and AWF [pQE16am-T415G / pRE] respectively Cells were cultured in M9 minimal medium with the addition of glucose, thiamine, MgSO4, CaCl₂, 20 amino acids (20 mg / L), antibiotics (kanamycin and ampicillin). When the cells reached the OD600 1.0 reading, they were pelleted by centrifugation, washed twice with cold 0.9% NaCl, and transferred to enriched M9 medium containing 17 amino acids (20 mg / L), 3mM unnatural amino acid of interest and the indicated concentration of Phe, Trp and Lys. Protein expression was induced by the addition of IPTG (1 mM). After 4 hours, the cells were pelleted and the proteins were purified using tegu C-terminal hexa Histidine and spin Nickel-NTA columns according to the manufacturer's instructions. (Qiagen).

EXAMPLE 6 [0351] The mDHFR mutant was purified under denaturing conditions and eluted with standard buffer (8 M urea, 100 mM Naf + PCFt, 10 mM Tris, pH 4.5). The polypeptides were digested with trypsin with 10 microliters diluted in 90 microliters 75 mM (NFLj + CCh and pH adjusted to 8. Two microliters of modified trypsin (0.2 micrograms / microliter) were added and samples incubated at room temperature overnight. The polypeptides were digested with Lys-C endoproteinase, 10 microliters diluted in 90 microliters 25 mM Tris-HCl, pH 8 and 1 mM EDTA. Then, 2 microliters of Lys-C (0.2 micrograms / microliter; CALBIOCHEM®) was added and the reaction mixture was incubated at 37 ° for 10 hours. The digestion reaction was stopped by adding 2 microliters of trifluoroacetic acid (TFA). The solution was purified with ZIPTIPcis® (MILLIPORE®) and digested peptides were eluted with 3 microliters 50% CH3CN, 0.1% TFA. One microliter was used for analysis by laser desorption mass spectrometry with matrix assisted ionization (MALDI-MS) with alpha-cyano-4-hydroxycinomonic acid and 2,5-dihydroxybenzoic acid as the matrix. The analysis was carried out using a PERSEPTIVE BIOSYSTEMS® Voyager DE PRO MALDI-TOF mass spectrometer in linear and positive ion modes.

[0352] LC-MS / MS analysis of protease digested peptides was performed by LCQ FINNIGAN® ion trap mass spectrometry with an HPLC pump and ESI probe. Tandem mass sequencing was performed by fragmenting the m / z precursor ions corresponding to the protease digested fragment containing the residue at position 38 of the mDHFR mutant.

EXAMPLE 7 [0353] Plasmids were constructed for wild-type yPheRS and yPheRS (T415G) as described in Example 1 herein. In addition, the lysS E.coli gene was amplified by PCR from the pXLLysKSl plasmid matrix using the following primers: 5'-GCA CTG ACC ATG GCT GAA CAA CAC GCA CAG-3 '(SEQ ID NO: 13) (with Acol restriction site) and -GGA CTT CGG ATC CTT TCT GTG GGC GCA TCG C-3 '(SEQ ID NO: 14) (with BamEIYy restriction site final hexa Histidine tags to facilitate protein purification.

[0354] In the first two reactions, (primer 1 and primer 4) and (primer 2 and primer 3) were added to individual tubes and two DNA fragments were generated from these two PCR reactions. From a mixture of two reaction products and other external primers, a 3400bp DNA fragment was obtained. The fragment was purified by standard methods and digested with BamH1 and Kpni and inserted into pQE32 giving pQE32-T415G. The cloned PheRS enzymes contained the N-terminal known sequence of hexa-histidine for purification. The entire yPheRS gene was subjected to DNA sequencing for verification.

EXAMPLE 8

[0355] Plasmid PQE32-T415A and pQE60-eLysS were individually co-transformed with the repressor plasmid pREP4 into the E. coli BLR strain to create BLR (pQE32-yFRS), BLR (pQE32-T415G), BLR (pQE32-TR15) expression strains (pQE60-Elysse). Overexpression was carried out in 2 χ YT 100 microgram / mL ampicillin and 35 microgram / mL kanamycin media. At OD 600 = 0.6, expression of the yPheRS variants and E. coli lysyl tRNA encoded by the lysS gene (eLysS) was induced with 1 mM IPTG. After 4 hours of expression, the cells were harvested and the proteins were purified on a nickel-nitrilotriacetic acid affinity column under native conditions according to the manufacturer's protocol (QIAGEN®). Imidazole in elution buffer was removed by desalting column and the polypeptides were eluted in buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM DTT. Polypeptide samples were stored at -80 ° C with 50% glycerol. Concentrations of yPheRS and eLysS variants were determined based on UV absorbance at 280 nm. EXAMPLE 9 [0356] Peptide 38 (residues 26-39; NGDLPWPPLRNEamber codonK) (SEQ ID NO: 15) contains the amber codon at position 38. Peptides (K38S, K38L), W38 peptide and pBrF (Z) 38 peptide were separated and detected by MS. Polypeptides were synthesized in triple auxotrophic host cells with (a) tRNA^phecuA and yPheRS (T415G); (b) tRNA^pheCuA_uG and yPheRS (T415G); (c) tRNA^pheCUA and yPheRS (T415A); (d) tRNA^pheCUA_uG and yPheRS (T415A) or (e) in a single auxotrophic strain with tRNA^phecuA_uo and yPheRS (T415A). Minimal expression medium was supplemented with 6.0 mM pBrF, 0.01 mM Trp, 1.0 mM Lys, 0.03 mM Phe (a and b) or 0.01 mM Phe (c, d and e) and 25 mg / L 17 amino acids, the results are shown in Figure 9.

EXAMPLE 10 [0357] The amino acid-dependent ATP-PP exchange reaction was used to evaluate the activation of amino acid analogs by yPheRS as described in the Examples above. Briefly, 200 microliter aliquots of reaction buffer contain 50 mM HEPES (pH 7.6), 20 nM MgCŁ, 1 mM DTT, 2 mM ATP, and 2mM³²P-pyrophosphate (PPi) with specific activity 10-50 Ci / mol. Depending on the activity of the analogues by the synthetase, the amino acid concentration ranged from 10 microM to 2.5 mM and the enzyme concentration ranged from 10nM to 100 nM. 20 microliter samples were removed from the reaction solution at various time points and the reaction was terminated by the addition of 500 microliters buffer solution containing 200 mN NaPPj, 7% w / v HclCfi, and 3% w / v activated carbon. The carbon was centrifuged and washed twice with 500 microliters of 10 mM NaPPj and 0.5% HClO4 solution. Radiolabeled ATP absorbed by carbon was quantified by liquid scintillation methods. Specificity constants were calculated by non-linear regression fit data for the Michaelis Menten model.

[0358] The results of the kinetic parameters are shown in Table I. Indole ring substitution (especially in the 6-position) was very favorable for some analogues (8-10).

Table I: Kinetic Parameters of ATP-PPi Exchange of Exemplary Amino Acids (1-11) by External Mutant Yeast PheRS.

<td>Amino acid</td><td>Enzyme</td><td>Κτη (μΜ)</td><td>kcat (s'¹)</td><td>kcat / Km (M ^ s'¹)</td><td>kcat / Km (relative) *</td>

<td> 1</td><td>T415G</td><td> 264+/-42</td><td> 0.05+/0.002</td><td> 184+/-30</td><td> 1</td>

<td> 2</td><td>T415G</td><td> 22+/-3</td><td> 0.03+/-0.001</td><td> 1,538+/-228</td><td> 8</td>

<td> 3</td><td>T415G</td><td> 12+/-2</td><td> 0.05+/-0.001</td><td> 4,365+/-797</td><td> 24</td>

<td> 4</td><td>T415G</td><td> 11+/-3</td><td> 0.05+/-0.002</td><td> 4,558+/-1,186</td><td> 25</td>

<td> 5</td><td>T415G</td><td> 757+/-149</td><td> 0.4+/-0.003</td><td> 48+/-10</td><td> 1\4</td>

<td> 6</td><td>T415G</td><td> 20+/-5</td><td> 0.30+/-0.006</td><td> 15,000+/-4,063</td><td> 82</td>

<td> 7</td><td>T415G</td><td> 27+/-2</td><td> 0.04+/-0.001</td><td> 1,550+/-125</td><td> 8</td>

<td> 8</td><td>T415G</td><td> 20+/-8</td><td> 0.20+/-0.018</td><td> 10,256+/-4,562</td><td> 56</td>

<td> 9</td><td>T415G</td><td> 8+/-4</td><td> 0.55+/-0.097</td><td> 70,876+/-34,843</td><td> 385</td>

<td> 10</td><td>T415G</td><td> 31+/-18</td><td> 0.06+/-0.005</td><td> 1,939+/-1,149</td><td> 10</td>

<td> 11</td><td>T415G</td><td> 94+/-50</td><td> 0.05+/-0.006</td><td> 533+/-293</td><td> 3</td>

<td> 1</td><td>T415G</td><td> 68+/-20</td><td> 0.52+/-0.093</td><td> 7.627+/-2.664</td><td> 41</td>

<td colspan="6">Where the amino acids are as in Figure 2.</td>

EXAMPLE 11 [0359] Mutated yeast suppressor amber tRNA (ytRNA^phecuA) was constitutively expressed under the control of the Ipp promoter. YtRNA expression cassette^phecuA was inserted into the repressor plasmid pREP4 to form pREP4-ytRNA as previously described in the Examples. Mutant yeast suppressor ytRNA^phecuA_30U40G (ytRNA^pheCuA ug) constructed from ytRNA^phecuA by using the QUICK-CHANGE® kit. Two complementary oligonucleotides, designated as primer UG-f (5'-GAA CAC AGG ACC TCC ACA TTT AGA GTA TGG CGC TCT CCC-3 ') (SEQ ID NO: 16) for forward primer and primer UG-r (5'- GGG AGA GCG CCA TAC TCT AAA TGT GGA GGT CCT GTG TTC-3 ') (SEQ ID NO: 17) for the reverse primer was synthesized to transfer a specific mutation to position 30 or position 40 of the mutated yeast suppressor tRNA. The resulting plasmid carrying the gene encoding ytRNA^phe cua_ug is marked as pREP4-ytRNA_UG. To construct a plasmid for in vitro transcription ytRNA^phe, ytRNA genes^pheCuA and ytRNA^phecuA ug was amplified from the plasmid matrix pREP4-ytRNA and pREP4-ytRNA_UG, respectively. At the end of the tRNA sequence, a BstNi site was inserted to generate an exact ytRNA transcript^phe. The T7 promoter sequence was added for in vitro transcription of ytRNA^phe by T7 RNA polymerase. The following primers were used for PCR: 5'-CTG GGT AAG CTT CGC TAA GGA TGA GCC CTG GTG CGA ACT CTG-3 '(SEQ ID NO: 18) (with HindIII and BstNi restriction sites) and 5'-GAT TAC GGA TTC CTA ATA CGA CTC ACT ATA GCG GAC TTA GCT C-923 '(SEQ ID NO: 19) (with EcoRI restriction site and T7 promoter sequence). The resulting DNA was introduced into HindIII / EcoRIpUC18 sites to give pUC18-ytRNA^pheCuA and pUC18-ytRNA^phecuA_UG · [0360] To facilitate DNA handling, one BstNI cleavage site near the T7 pUC18-ytRNA T7 promoter sequence^phecuA was removed to increase the size of the DNA fragment containing the ytRNA gene^phecuA from 180 bp to 500 bp after digestion with BstNI. Two complementary oligonucleotides, 5-CGG AAG CAG AAA GTG TAA AGA GCG GGG TGC CTA ATG AGT G-3 '(SEQ ID NO: 20) for forward primer and 5'-CAC TCA TTA GGC ACC CCG CTC TTT ACA CTT TAT GCT TCC G-3 '(SEQ ID NO: 21) for the reverse primer, was synthesized to carry a specific mutation.

EXAMPLE 12 [0361] Linearized DNA was prepared by digesting BstNI pUC18-ytRNA^phec_UA and pUC18-ytRNA^phecuA_uG as previously described (see Sampson, Uhlenbeck, PNAS USA 85: 1033-1037 (1988)). In vitro transcription of linearized DNA templates and purification of transcripts was performed as previously described (see Nowak, et al., Channels Channels B 293: 504-529 (1998). In vitro transcription of linearized DNA to generate 76mer tRNA transcripts was performed using a kit T7MEGASHORTSCRIPT® AMBION®. The transcripts were isolated by extraction 25: 24: 1 phenol: CHCfi: isoamyl alcohol (PCI). The organic layer was re-extracted with water and 24: 1 CHCl isoamyl alcohol (CI) extraction was performed on the aqueous layers. The aqueous layer was then mixed with an equal volume of isopropanol, precipitated overnight at -20 ° C, deposited, dried and redissolved in water. Unreacted nucleotides in the tRNA solution were eliminated using CHROMA SPIN-30® DEPC-H2O (BD Bioscience®). Transcript concentrations were determined based on UV absorbance at 260 nm.

[0362] Aminoacylation of wild type ytRNA^pheGAA using Phe and Trp via yPheRS variants was performed as described previously (see Sampson, Uhlenbeck, PNAS USA 85: 1033-1037 (1988)). Aminoacylation reactions were carried out in a buffer containing 30 mM HEPES (pH 7.45), 15 mM MgCl2, 4mM DTT, 25mM KCl, and 2mM ATP at 30 ° C, in 100 microliters of reaction volume. Purified total yeast tRNA was used in the assay at a final concentration of 4 mg / mL (ytRNA concentration^pheGAA approximately 2.24 microM). For aminoacylation of Phe, variants 13.3 microM [H] -Phe (5.3 Ci / mmol) and 80 nM yPheRS were used; for aminoacylation of Trp, variants 3.3 microM were used [³H] -Trp (30.0 Ci / mmol) and 160 nM yPheRS. Aminoacylation of ytRNA transcripts^phewas carried out in 100 microliters of reaction volume in a buffer containing 100 mM potassium-HEPES (pH 7.4), 10 mM MgCl2, 1 mM DTT, 0.2 mM EDTA, 2 mM ATPi 4 units / mL yeast inorganic pyrophosphatase at 37 ° C for eLysS. For aminoacylation of Lys, 4 microM ytRNA digest was used^phe, 1.1 microM [³H] -Lys (91 Ci / mmol) and 80 nM eLysS. tRNA was fused prior to use by heating to 85 ° C for 4 minutes in fusion buffer (60 mM Tris, pH 7.8, 2 mM MgClf) and then slowly cooled to room temperature. Reactions were initiated by the addition of enzyme and 10

-93 microliter portions were quenched by applying a spot to Whatman filter discs impregnated with 5% TCA. Filters were washed for three 10 minutes in ice-cold 5% TCA, washed with ice-cold 95% ethanol, and counted by liquid scintillation methods.

EXAMPLE 13 [0363] Construction of the plasmid for in vivo introduction of an unnatural amino acid was carried out in the double auxotrophic Phe / Trp, AFW strain and the triple auxotrophic Phe / Trp / Lys, AFWK strain. These auxotrophic strains were constructed from the auxotrophic strain Phe, AF (KIO, Hfr (Cavalli) pheS13rel-1 tonA22 thi T2^R/? / zeA18, trpBY \ A) (see Furter, Prot. Sci, 7: 419-426 (1998)) by PI-phage mediated transposon transduction. The pQE16 vector (QIAGEN®) was selected as the expression plasmid that codes for the mouse dihydrofolate reductase (mDHFR) marker protein with the C-terminal hexa-histidine tag gene under the control of the bacteriophage T5 promoter and t terminator₀. The Quick-change mutagenesis kit was used to place the amber codon (TAG) at position 38. mDHFR with two complementary oligonucleotides (5'-CCG CTC AGG AAC GAG TAG AAG TAC TTC CAA AGA ATG-3 '(SEQ ID NO: 11); 5'-CAT TCT TTG GAA GTA CTT CTA CTC GTT CCT GAG CGG-3 ') (SEQ ID NO: 12) giving pQE16am. The mutated yPheRS genes T415G and T415A were amplified from pQE32-T415G and pQE32-T415A and the constitutive tac promoter with the lac repressor binding site removed was added upstream of the gene start codon. The entire T415G and T415A expression cassette was placed into the Pvull site in pQE16am-T415G and pQE16am-T415A.

EXAMPLE 14 [0364] These auxotrophic bacterial strains AF, AFW and AFWK were transformed with plasmid pQE16am containing yPheRS variants and vectors, pREP4-ytRNA containing ytRNA variants to examine the introduction of pBrF. E.coli expression strains were cultured in minimal M9 medium supplemented with glucose, thiamine, MgSCfi, CaCŁ, 20 amino acids (at 25 mg / L) with antibiotics (35 micrograms / mL kanamycin and 100 micrograms / mL ampicillin). When the cells have reached OD₆oo 0.8-1.0, pelleted by centrifugation, washed twice with cold 0.9% NaCl, and transferred to expression medium supplemented with 17 amino acids (at 20 mg / L), 6 mM pBrF (p-bromo-phenylalanine) or pJodoF (p-iodoF phenylalanine), and indicated concentrations of phenylalanine, tryptophan and lysine. Protein expression was induced by the addition of ImM IPTG. After four hours of expression, the cells were pelleted by centrifugation, and the protein was purified thanks to the C-terminal tag of hexa-histidine by a -NTA spin-nickel NTA column according to the manufacturer's instructions (QIAGEN®). After purification, mDHFR expression level was determined by measuring UV absorbance at 280 nm.

EXAMPLE 15 [0365] LC-MS / MS analysis of tryptically digested peptides was performed with LCQ FINNIGAN® ion trap mass spectrometry with an HPLC pump and ESI probe. The mDHFR mutant was purified under denaturing conditions in elution buffer (8M

- urea, 100 mM Nal + PCU, 10 mM Tris, pH 4.5). For trypsin digestion, 10 microL solution was diluted to 90 microL 75 mM (Nl + fiCCfi. One microliter of modified trypsin (0.2 micrograms / microliter) was added. The sample was incubated at 37 ° C for 2 to 6 hours. The digestion reaction was stopped by adding 12 microL % TFA solution Digested peptide was desalted using a 08 Vydac Microspin column (Nest group) and 50 microL 80% acetonitrile and 20% 0.1% w / v formic acid were eluted. The digested peptide solution eluted from the Microspin column was dried, redissolved in 10% acetonitrile and 90% 0.1% TFA, and injected into the HPLC pump. The peptides were separated by a Magie C18 column (Michrom, 300 A, 0.3 x 150 mm) and eluted at a flow rate of 30 microL / min using a gradient of 10-95% solvent A (90% acetonitrile and 10% 0.1 M acetic acid solution) and solvent B (2% acetonitrile and 98% 0.1 M acetic acid solution) for 30 minutes. The column eluent was directed to an electrospray source and each signal from trypsin digestion was detected. Tandem mass sequencing was carried out simultaneously by fragmentation of the m / z precursor ions corresponding to the protease digested fragment containing the residue at position 38 of the mDHFR mutant. Thus, DHFR polypeptides were synthesized in triple auxotrophic host cells (a), (b) yeast tRNA^phecuA and yeast PheRS (T415G); (c) yeast tRNA^phecuA_uG and yeast PheRS (T415G); (d) yeast tRNA^phecuA and yeast PheRS (T415A); (e) yeast tRNA^pheCUA-uG and yeast PheRS (T415A) or (f) in an individually auxotrophic yeast tRNA strain^BCcuA_uG and yeast PheRS (T415A), as shown in Figure 12.

EXAMPLE 17 [0366] The yeast PheRS library (using green fluorescent protein or GFP) was screened to identify PheRS mutations that allow the introduction of Nal. Individual amino acid sequence positions that have been mutated include residue numbers 412, 415, 418 and 437, which are located at the binding site and are in contact with the amino acids. As indicated in Figure 13, p-ethynyl-phenylalanine was introduced into the test protein using modified yeast PheRS.

[0367] Briefly, GFP was ligated into a vector containing the mutated T415G or wild-type yeast PheRS gene according to standard procedures. Mutated yeast suppressor amber tRNA (ytRNA^pheAAA) was constitutively expressed under the control of the Ipp promoter and transformed into the double auxotrophic E.coli AFW Phe / Trp strain. Cells were cultured in M9 minimal medium supplemented with glucose, thiamine, MgSO₄, CaCf, 20 amino acids (at 25 mg / L), antibiotics (35 pg / mL kanamycin and 100 pg / mL ampicillin). When the cells reached ODo 0.8-1.0, the cells were pelleted and washed twice with ice-cold 0.9% NaCl and transferred to expression medium supplemented with 18 amino acids (at 20 mg / L) and various concentrations of phenylalanine, tryptophan and 2Nal. Protein expression was induced by IPTG.

EXAMPLE 18

[0368] Mutagenesis of selected four amino acid residues (N412, T145, S418, and S437) was performed according to a two-step PCR mutation. Briefly, a series of PCR mutagenesis was performed on the GFPuv gene in plasmid pQE9_GFPuv (STRATAGENE®), using four pairs of complementary primers (F64LS65T_f: 5-CTT GTC ACT ACT CTG ACC TAT GGT GTT CAA TGC TTC CQT NO: ' 22); F64LS65T_r: 5'ACG GGA GAA GCA TTG AAC ACC ATA GGT CAG AGT AGT GAC AAG-3 '(SEQ IDNO: 23); S99Ff: 5'-GTA CAG GAA CGC ACT ATA TTC TTC AAA GAT GAC GGG AAC- 3 '(SEQ ID NO: 24); S99F_r: 5'-GTT CCC GTC ATC TTT GAA GAA TAT AGT GCG TTC CTG TAC-3' (SEQ ID NO: 25); Τ153Μ Τ 5'- CAC AAT GTA TAC ATC ATG GCA GAC AAA CAA AAG AAT GGA-3 '(SEQ ID NO: 26); T153M_r: 5'-TCC ATT CTT TTG TTT GTC TGC CAT GAT GTA TAC ATT GTG -3 '(SEQ ID NO: 27)).

[0369] GFP mutants were prepared as described herein. In short, GFP3 has 12 Phe residues of which five are encoded by Phe wobble codons (UUU). The GFP5 and GFP6 variants were generated by replacing UUC codons with UUU codons by a two-step ligation-ended PCR. GFP5 was produced by replacing four UUC codons and one Leu codon at residues F8, L64, F84, F99 and FI 65 with codons UUU using twelve primers (1: 5'-GTG CCA CCT GAC GTC TAA GAA ACC ATT ATT ATC ATG ACA TTA ACC -3 '(SEQ ID NO: 28) 2: 5'-GAG TAA AGG AGA AGA ACT TTT TAC TGG AGT TGT CCC AAT TC-3' (SEQ ID NO: 29) 3: 5'- GAA TTG GGA CAA CTC CAG TAA AAA GTT CTT CTC CTT TAC TC-3 '(SEQ ID NO: 30) 4: 5'- GGC CAA CAC TTG TCA CTA CTT TTA CCT ATG GTG TTC AAT GCT T-3' (SEQ ID NO: 31) 5: 5'- AAG CAT TGA ACA CCA TAG GTA AAA GTA GTG ACA AGT GTT GGC C3 '(SEQ ID NO: 32) 6: 5'- CAT ATG AAA CGG CAT GAC TTT TTT AAG AGT GCC ATG CCC GAA G -3 '(SEQ ID NO: 33) 7: 5'- CTT CGG GCA TGG CAC TCT TAA AAA AGT CAT GCC GTT TCA TAT G (SEQ ID NO: 34) 8: 5'- GTT ATG TAC AGG AAC GCA CTA TAT TTT TCA AAG ATG ACG GGA ACT ACA A-3 '(SEQ ID NO: 35) 9: 5'- TTG TAG TTC CCG TCA TCT TTG AAA AAT ATA GTG CGT TCC TGT ACA TAA C-3' (SEQ ID NO: 36 ) 10: 5'- ACA AAA GAA TGG AAT CAA AGC TAA CTT TAA AAT TCG CCA CAA CAT TGA AGA TG-3 '(SEQ ID NO: 37) 11: 5'- CAT CTT CAA TGT TGT GGC GAA TTT TAA AGT TAG CTT TGA TTC CAT TCT TTT GT-3 '(SEQ ID NO: 38); 12: 5'- CGC CAA GCT AGC TTG GAT TCT CAC CAA TAA AAA ACG CCC-3 '(SEQ ID NO: 39) Five partially overlapping fragments of GFP3 expression cassettes were obtained by five PCR reactions with five sets of primers (1 and 3 ; 2 and 5; 4 and 7; 6 and 9; 8 and 10).

[0370] These PCR reaction products were purified by agarose gel electrophoresis followed by gel extraction. GFP6, whose all Phe residues are encoded by UUU, was generated by replacing two Phe codons (F71 and F99) with GFP5 codons with UUU using six primers. (1 to 12 are the same as above; 13: 5'- TAC CTA TGG TGT TCA ATG CTT TTC CCG TTA TCC GGA TCA TAT G-3 '(SEQ ID NO: 40); 14: 5'-CAT ATG ATC CGG ATA ACG GGA AAA GCA TTG AAC ACC ATA GGT A-3 '(SEQ ID NO: 41); 15: 5'- GTT ATG TAC AGG AAC GCA CTA TAT TTT TTA AAG ATG ACG GGA ACT ACA AG-3' (SEQ ID NO: 42); 16: 5'- CTT GTA GTT CCC GTC ATC TTT AAA AAA TAT AGT GCG TTC CTG TAC ATA AC-3 '(SEQ ID NO: 43)).

EXAMPLE 19 [0371] The library was also built in two stages. Briefly, saturation mutation at four residues (N412, T415, S418 and S437) was performed by two-step PCR mutagenesis. First, degenerate codons were introduced into S437 by PCR mutagenesis with two complementary primers (437 f: 5'-GTC GAA ATC GGT AAC NNK GGT ATG TTC AGA CCA GAA ATG CTC G3 '(SEQ ID NO: 44); 437_r: 5 '- C GAG CAT TTC TGG TCT GAA CAT ACC MNN GTT AC C GAT TTC GAC-3' (SEQ ID NO: 45)). After digestion 1 hour. PCR product using Dpni, the PCR product was transformed into an XL-1 Blue cloning host. Plasmids at position 437. were saturated and isolated and used as template for 2. PCR mutagenesis to introduce mutations of residues N412, T415 and S418. 2. PCR mutagenesis was performed with another complementary pair of primers (412 418_f: 5'-C AAG CCT ACC TAC NNK CCT TAC NNK GAG CCA NNK ATG GAA ATC TTT T-3 * (SEQ ID NO: 46); 412_418_r: 5'-A AAA GAT TTC CAT MNN TGG CTC MNN GTA AGG MN N GTA GG T AGG CTT G-3 '(SEQ ID NO: 47)). After PCR, products were digested with Dpn1 for 1 hr, cleaned and concentrated using a spin column. The eluate was electroporated into ElectroTen Blue electrocompetent cells (Stratagene) according to the manufacturer's protocol. Eight million transformants were received. The plasmid library was expanded in culture and digested with Nsi \ and BglR. After purification of these inserts, they are ligated with large fragments pQE9_GFP6_yPheRS (T415G) and pQE9 GFP9 yPheRS (T415G) obtained by digestion with Nsi1 and Bglli.

[0372] The library was transformed into chemically competent E. coli AFW and DHF cells. These cells were then inoculated into 2 χ YT media with kanamycin and cultured overnight. When the cells reached 0-8 OD8, ​​the cells were pelleted and resuspended in distilled water. Stock glycerin solutions of the library were expressed as is known in the art.

[0373] After GFP expression within 3 hours, 1 mL cells (based on OD₆₀1.0) was washed with PBS and diluted with distilled water and then subjected to flow cytometry analysis (MoFlo cell sorter®, DakoCytomation, Fort Collins, CO) at 488 nm excitation wavelength, 525 nm emission, and a 495 nm cut-off filter. At least 20,000 events were collected in each measurement. Data were analyzed with Summit software (DakoCytomation). The library was screened both positively and negatively, i.e., yPheRS variants that allow high introduction of 2Nal or other natural amino acid except for Phe at the UUU codons, will develop GFP and become less bright and thus low fluorescence cells are harvested. The yPheRS variants that do not allow the introduction of other natural amino acids except Phe at the UUU codons, do not affect GFP folding and are clear. So bright cells are collected. Figure 14 shows histograms of screening the yPheRS library from GFP.

SEQUENCE LIST [0374]

-97 &lt;110&gt; Califomia Institute of Technology

Wang, Pin

Kwon, Inchan

Son, Soojin

Tang, Yi

Tirrell, David A.

&lt;120&gt; LOCAL SPECIFIC ENTERING OF AMINO ACIDS IN

PARTICLE &lt;130&gt; 110197 412PC &lt;140&gt; PCT &lt;141&gt; 2007-03-05 &lt;150&gt; US 60 / 779,375 &lt;151&gt; 2006-03-03 &lt;150&gt; US 60 / 779,376 &lt;151&gt; 2006-03-03 &lt;160 &gt; 104 &lt;170&gt; FastSEQ for Windows Version 4.0 &lt;210&gt; 1 &lt;211&gt; 7790 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Modified mouse dihydrofolate reductase gene and E. coli MetRS gene. &lt;400&gt; 1

-98ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca 60 actgtgagcg gacaacaatt tcacacagaa ttcattaaag aggagaaatt aactatgaga 120 ggatcgcatc accatcacca tcacggatcc ggcatcatgg ttcgaccatt gaactcgatc 180 gtcgccgtgt cccaaaatat ggggattggc aagaacggag acctaccctg gcctccgctc 240 aggaacgagt tcaagtactt ccaaagaatg accacaacct cttcagtgga aggtaaacag 300 aatctggtga ttatgggtag gaaaacctgg ttctccattc ctgagaagaa tcgaccttta 360 aaggacagaa ttaatatagt tctcagtaga gaactcaaag aaccaccacg aggagctcat 420 tttcttgcca aaagtttgga tgatgcctta agacttattg aacaaccgga attggcaagt 480 aaagtagaca tggtttggat agtcggaggc agttctgttt accaggaagc catgaatcaa 540 ccaggccacc ttagactctt tgtgacaagg atcatgcagg aatttgaaag tgacacgttt 600 ttcccagaaa ttgatttggg gaaatataaa cttctcccag aatacccagg cgtcctctct 660 gaggtccagg aggaaaaagg catcaagtat aagtttgaag tctacgagaa gaaaggttgg 720 aagatcttaa gcttaattag ctgagcttgg actcctgttg atagatccag taatgacctc 790 agaactccat ctggatttgt tcagaacgct cggttgccgc cgggcgtttt ttattggtga 840 gaatccaagc tagctctaga gacgtccggc cggagctcca ccgcggtggc ggccgctcta 900 gagtcactta cttaacattt tcccatttgg tactatctaa ccccttttca ctattaagaa 960 gtaatgccta ctatgactca agtcgcgaag aaaattctgg tgacgtgcgc actgccgtac 1020 gctaacggct caatccacct cggccatatg ctggagcaca tccaggctga tgtctgggtc 1080 cgttaccagc gaatgcgcgg ccacgaggtc aacttcatct gcgccgacga tgcccacggt 1140 acaccgatca tgctgaaagc tcagcagctt ggtatcaccc cggagcagat gattggcgaa 1200

-99atgagccagg

Lcgacgcaca aacggtttta ctgccggacc gacaactgcg gtggtttctg tctttcagcg aataaaatgc ecttacttcg gcaccgatcg agcttcgatg gatattgttt aagccgtcca tctcgcggca cgttactact gatttcgctc aatgcgggct cagttgtaca gaatttggta gatgaacagg atttgctcaa ccgaaactga cagcaaccgc atgaggcagg ccggtaactg gctaaagttg aaactgctgc cgttctgctt ctggcaceac ggcgggaaag aaataatccc gttttcttgc ctggataaaa taaattcagg caccaaaggc cccgacctcg ttttcaggag acatcccaat tataaccaga cacaagtttt tttcgtatgg aecgttttcc ttccggcagt tattecccta ttcaccagtt atgggcaaat catgccgttt gatgagtggc ggggtaatga ggcctttcgt ctctagatca agctaactta tgccagccgc cagggtggtt gccctgagag tttgatggfcg taccgagaca cgccatctga agcatcagac gcgaagagaa ttaaaaaccg gttttgtgaa aagtctgegg gcgctacgcc aaatgttgca aggagtggtt gttttgaaat gctacatggg aatactggaa acttccacag acctgtttgt cctttattaa acacCgcgaa agcgtgngaa ttatcaacaa aaacccccac aagccgtgcg ctccgcgggt tgggcatcaa ccgagcgtgc tgetgggcca ttgaagcact gcccgctggc aectgcgcgt gcctgacgct acccggaCcc gtaaaatgcg atattttcct ccttcaaggc gattttgtct ttacagggat gcagttgagc aaagccagat agggggggcc ctaaggaagc ggcatcgtaa ccgttcagct atecggcctt caatgaaaga atgagcaaac ttctacacat aagggtttat ttgatttaaa attatacgca gtgatggctt agggcggggc ctctctajct tttatctgtt cgtgcagtcg cattaattgc attaatgaat tttcttteca agttgcagca gteaacggcg tccgcaccaa tcgttggcaa tgatttcgca ccgccagti string caccatctct aggcacctgc cgcgacctac ggtaatgcgt ggcatggacc tgaatctggc tccgaacgcg ttctttcaag gaaagactcc cctgttctgg tcacggctat agccagcacc actctcttcg tgccgatatc gcgttttgac tgatgccgct cgaaatcatg ggtggcgaaa cctgttccgc agaagcattc caaagtgaat ggtggaagcc agatgatccg ggcgctgatt ggatctcggc gcaggcactg cttcggtatc gctaagcccg gctgcatcga ctctctaacc gcagaatgag aacagggaaa aaaaagtttt cggtacccgg taaaatggag agaacatttc ggatattacg tattcacatt cggtgagctg tgaaacgttt atattcgcaa tgagaatatg cgtggccaat aggcgacaag ccatgtcggc gcaatttttt tgaggcatca gtttgtcggt atgacaagct gttgcgctca cggccaacgc ccagtgagac agcggcccae ggatataaca cgcgcagccc ccagcaccgc ggctttaaca tcagaactta cagctgtacg ccgaaatgta agcccgactg gattctgaac cgcagcggtg ctgcaacagt ccgggcaaat aatctgtgcg accgccgagc cctgccatgc gtgacggcga tggctgaatc cgcattgatg gctaacaaag ggcgtgctgg gaagcgattg gcgctggctg caggaaggcc gtgctgatga ctcaatacgg ccgttcaagg tctaaatgag attcaggaaa gaaaacgcag ggtgaaaaac attggtcgtc tctgaaggca gatgccggtg cagccttttg egcataaata acactttatc cgagttgcat tcagatgctg ccggacgtct aaaaaaacca gaggcatttc gcctttttaa cttgcccgcc gtgatatggg tcatcgctct gatgtggcgt tttttcgtct atggacaact gtgctgatgc agaatgctta taaggcagtt aataaaacga gaacgctctc gtcaaacatg ctgcccgctt gcggggagag gggcaacagc gctggtttgc tgagctgtct ggactcggta agtgggaacg tcagctatga tctactctcg atccggaaaa aatccccgga aactgatcga acttcttctt cgttgcagga gggatatctc atttctacgt acaagcgcgg tgtaccactt tggaaggcag acggcgcaaa attttgacgc atatcgatct tggttaacct caagcgaact gtgaagcgcg atctggctaa gcgatgccga cttacctgaa aactgacctg cgctgtataa aagcaaaagc ccatcacctt agtttgttga gcaatgtctt acaccattat tggtgatggc ccaaaccggg ctttataaat ctggtagcat tatcaggacg ateagttggg cggcagcggc ctagagctag ctggatatac aqtcagttgc agaccgtaaa tgatgaatgc atagtgttca ggagtgaata gttacggtga cagccaatcc tcctcgcecc cgctggcgat atgaattaca attggtgccc aaggctcagt ctgagtagga agaattgtgc tccagtcggg gcggtttgcg cgattgccct cccagcaggc tcggtatcjt atggcgcgca atgccctcat caactatcac 1260 cctgaaagaa 1320 aggcatgttc 1380 tcaatacggc 1440 gccgaaatcg 1500 tgatctgccc 1560 geaggtggca 1620 ccgcgacgcc 1660 ctggctggac 1740 cgacagcgta 1800 catcggtaaa 1860 caacttccgc 1920 gatgcccaag 1980 agacagcctg 2040 caacctggaa 2100 ggcctcccgt 2160 ggctgacccg 2220 ggaaagccgt 2280 ccgctatgtc 2340 cctgcaggca 2400 gccggtactg 2460 ggatggtatc 2520 ccgcatcgat 2530 cgctgccgcg 2640 tgacgacttc 2700 aggttctgac 2760 ctccggtatt 2820 ggtggcbaac 2380 tgccggtcct 2940 tcatcaggtg 3000 tcctaaagtt 3060 ctgcattcaa 3120 aaaaatcaca 3180 gaaaagttgg 3240 aaaagcggag 3300 ettggcgaga 3360 caecgttgat 3420 tcaatgtacc 3480 gaaaaataag 3540 tcatccggaa 3600 cccttgttac 3660 ccacgacgat 3720 aaacctggcc 3730 ctgggtgagt 3840 cgttttcaec 3900 tcaggttcat 3960 acagtactgc 4020 ttaaacgcct 4060 cgaaagactg 4140 caaatccgcc 4200 ctaatgagtg 4260 aaacctgtcg 4320 tategggcgc 4 3 80 tcaccgcctg 4440 gaaaatcctg 4500 cgtatcccac 4560 ttgcgcccag 4620 tcagcatttg 4680

-100catggttcgt tgaaaaccgg acatggcact ccagtcgcct tcccgttcęg ctatcggctg 4740 aatttgattg cgagcgagac atttatgcca gccagccaga cgcagacgcg ccgagaeaga 4800 acttaatggg cccgctaaca gcgcgattng ctggtgaccc aatgcgacca gatgctccac 4880 gcccagtcgc gtaecgtctt catgggagaa aaeaatactg ttgatgggtg tctggtcaga 4920 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg 4980 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac 5040 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc 5100 cagttgatcg gcgcgagatt taategccgc gacaatttgc gacggcgegt gcagggccag 5160 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg 5220 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcecgcg ttttcgeaga 5280 aacgtggctg gcctggttca ccacgcggga aacggtctga Łaagagacac cggcatactc 5340 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg 5400 gcgctatcat gccataccgc gaaaggtttt gcaceacccg atggtgtcgg aatttegggc 5460 agcgttgggt cctggccacg ggtgcgcatg acctagagct gccccgcgcg tttcggtgat 5520 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg 5580 gaegccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc 5640 gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat 5700 cagagcagat tgtactgaga gtgeaccata tgcggtgtga aataccgcac agatgcgtaa 5760 ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 5820 tcgttcggct gcggcgagcg gtabcagctc actcaaaggc ggtaatacgg ttatccacag 5880 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 5940 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 6000 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgc 6060 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 6120 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 6180 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgtteage 6240 ccgaccgetg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 6300 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 6360 ctacagagtt cttgaagtgg tggcctaact acggctacac fcagaaggaca gtatttggta 6420 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 6480 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 6540 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 6600 aaaactcacg ctaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 6660 ttttaaatta aaaatgaagt tttaaatcaa tcCaaagtat atatgagtaa acttggtctg 6720 acagttacca atgcttaatc agtgaggcac ccatctcagc gatctgtcta tttcgttcat 6780 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 6840 gccceagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagae ttatcagcaa 6900 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 6960 tccagtctat taattgttgc cgggaagcta gagcaagtag ttegecagtt aatagtttgc 7020 gcaacgfctgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 7080 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 7140 aagcggetag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 7200 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 7260 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 7320 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 7380 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 7440 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 7500 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 7560 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 7620 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 7680 gggttccgcg cacatetccc cgaaaagtgc cacctgacgt ctaagaaacc attactatca 7740 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcac 7790 &lt;210&gt; 2 &lt;211&gt; 8294 &lt;212&gt; DNA &lt;213&gt; yeast phenylalanine aminoacyl synthetase gene &lt;400&gt; 2

-101ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca 60 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aactatgaga 120 ggatcgcatc accatcacca tcacggatcc gtcgacctgc agccccgggt accggtagaa 180 aaaatgagta aaggagaaga actttttact ggagttgtcc caattcttgt tgaattagat 240 ggtgatgtta atgggcacaa attttctgtc agtggagagg gtgaaggtga tgcaacatac 300 ggaaaactta cccttaaatt tatttgcact actggaaaac tacctgttcc atggccaaca 360 cttgtcacta cttttaccca tggcgttcaa tgcttttccc gttatccgga tcatatgaaa 420 cggcatgact tttttaagag cgccatgccc gaaggttatg cacaggaacg cactatatct 480 tttaaagatg acgggaacta caagacgcgt gctgaagtca agtttgaagg tgataccctt 540 gttaatcgta tcgagttaaa aggtattgat tttaaagaag atggaaacat tctcggacac 600 aaactcgagt acaactataa ctcacacaat gtatacatca tggcagacaa acaaaagaat 660 ggaatcaaag ctaactttaa aattcgccac aacattgaag atggatccgt tcaactagca 720 gaccattate aacaaaatac cccaattggc gatggccctg tccttttacc agacaaccat 780 tacctgtcga cacaatctgc cctttcgaaa gatcccaacg aaaagcgtga ccacatggtc 840 cttcttgagt ttgtaactgc cgctgggatt acacatggca tggatgagct ctacaaactg 900 cagccaagct taattagctg aagcttggac tcctgttgat agatccagta atgacctcag 960 aactccatct ggatttgttc agaacgctcg gntgccgccg ggcgtttttt attggtgaga 1020 atccaagcta gcttggcgag attttcagga gctaaggaag ctaaaatgga gaaaaaaatc 1080 actggatata ccaccgttga tatatcccaa tggcatcgca aagaacattt tgaggcattt 1140 cagtcagttg ctcaatgtac ctataaccag accgttcagc tggcacgaca ggtttcccga 1200 etggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc 1260 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca 1320 atttcacaca ggaaacagct atgaccatga ttacgccaag cttgcatgcc tgcagttgac 1380 aattaatcat cggctcgtat aatggatcca attgtgagcg gaatcgattt tcacacagga 1440 aacagaccat gaatctagag atgtctgact tccaattaga aattctaaag aaactagatg 1500 aattggatga gatcaagtcc acactggcaa ctttccctca gcaćggctct caagatgttc 1560 tttccgcttt gaactctttg aaagcccaca acaagttaga gttttccaag gncgacacgg 1620 ttacgtatga cttgaccaaa gaaggtgctc aaattttgaa tgaaggttcg tacgaaatta 1680 aactagtcaa gctcatccaa gagttgggtc aacttcaaat caaagatgtg atgtccaaac 1740 taggccctca agttggtaag gtcggtcagg ctagagcttt caagaacggc tggatcgcca 1800 aaaacgcctc aaacgagctt gaactctccg caaaattgca aaataccgat ttaaatgagc 1860 ttaccgatga aacgcaatct attctagcgc aaatcaagaa caactcgcat ctggatagca 1920 Łtgacgccaa gattttgaac gacttgaaga aaagaaagtt aattgctcaa ggtaaaatca 1980 cagatttcag tgtcaccaaa gggccagagt tctcgaccga cctcaceaaa ttggaaaccg 2040 atcttacctc cgacatggtc tccaccaatg catacaagga cttgaagttc aagccttaca 2100 atttcaattc tcaaggtgtg caaatatctt caggtgctct tcacccctta aacaaagtca 2160 gagaggaatt tagacaaatt ttcttttcca tgggattcac agagatgccc tcgaaccaat 2220 acgtcgagac aggtttctgg aacttcgatg ccctttacgt cccacaacag catcctgctc 2280 gtgacctgca agacactttc tacatcaagg acccactaac cgctgagttg cccgatgaca 2340 agacatacat ggacaatatc aaagccgttc acgaacaggg gagattcggg tccatcggtt 2400 atcgttacaa ctggaagcca gaagaatgtc aaaaattggt cttgagaact cactccacag 2460 ccatctctgc cagaatgctg cacgatttgg ccaaagatcc aaagcccacc agattgtttt 2520 ctatcgaccg tgttttccgt aacgaagcag ttgacgccac ccatttggcc gaattccacc 2580 aggtggaagg tgttcttgcc gactacaaca ttactctggg tgacctgatc aagttcatgg 2640 aagagttctc cgaaagaacg ggtgtcaccg gttcgagatt caagcctacc tacaatcctt 2700 acaccgagcc atcaatggaa atcttttctt ggcacgaagg tttgcaaaaa tgggtcgaaa 2760 tcggtaactc tggtatgttc agaccagaaa tgctcgagtc catgggtcta ccaaaggatc 2820 taagagtcct tggttggggg ttatccttgg aaagacctac catgatcaaa tataaggttc 2880 aaaacatcag agaactgtta ggtcataaag tctctttgga ctttatcgaa accaatcctg 2940 ctgctagatt ggacgaagac ttgtacgaat aaggcaggaa tagattatgc ctaccgtctc 3000 cgtgaacaag cagcaattat ttgatcttct aggcaaagac tacacttccc aagagttcga 3060 cgaattatgt tttgaattcg gtatggaaat ggacgaagac accacagaag aggccttgaa 3120 aaccggggag gagccggaat tgaagcttga tatcagtgcc aatcgttaęg atttgctttg 3180 tatcgaaggt atttcacagt cgctgaacga atacttggaa cgtaaagaaa gacctgacta 3240 taaactaagc aagccaacca ctaagttgat catcgacaaa tcaacggagc aaattagacc 3300 ttttgctacc gctgctgtat tgagaaatat caagcttaac gaaaaatctt acgcttcttt 3360 tattgccttg caagataaat tacatgccaa tccatgtaga aacagaagct cggttgccat 3420

-102gggtactcac gatttagatt caattgaagg tccattccat tacagagctc taccaccaaa 3480 ggacatcaag ttcgtaccat tgaatcaaac ccaagagttt actggtgaća aattgatcga 3540 gttttataaa tctccagaac agaaaaacaa catagggaga tacgttcaca ttattgagga 3600 ttctccagtc tccccagtta ttacggacag caaagatcgt gtttgctccc tgccaccatt 3660 aatcaatagt gaacattcga agatctctgt gaacacccgt aacattttga ttgatataac 3720 cgccaccgat aagaccaaag ccgagatcgt tttgaaeata ttaactacaa tgttctcacg 3780 ttattgtgac gaaccattca cggttgagcc tgtagaaatt gtctctgaac acaatggcca 3840 atcccgtttg gcgccaaact tcaacgatag aattatggat gtctccatta agtacatcaa 3900 ctcctgtctt ggcctagatc aatccgctga tgaaattgct cattgtctaa aaaagatgtc 3960 gttgcatgcc gttcaatcaa aggaagacaa ggacatcttg cacgttgaca ttccggtaac 4020 tagacctgat attttgcacg ct tgtgatat aatggaagat gccgctgtcg gttatggttt 40B0 caataatttg ccaaagggtg agaaattatc caatgccaac ttcattgcca aaccattacc 4140 aatcaacaag gtttctgata ttttcagagt tgcatcctct caagccacgt gggttgaggt 4200 tttaccattg accttatgtt cgcacgatga aaactttaaa tttctaagac aatccgacaa 4260 tgatgattta gctgtcaaat tggccaaccc aaagactttg gaataccaag ttgctagaac 4320 cactttattg cctggtatct taaagacagt caaggaaaac agaaaacatt ccttaccaat 4380 caaagtcttt gaaaccggtg acgttgtatt taaagacgac aaactagaaa ggaaggcgta 4440 caatgaacgt cactgggctg ccatctacgt gggtaagaat tctgggtttg aaatcattca 4500 agggttattg ggtaaaatca tgcaaacttt tagaacagag tggattgcag actacggtgc 4560 tgctgcttct ggcagaggtt actggattga agaagacgat tctgtgaaaa cctatttccc 4620 aggtagaggt gccaaggtca tgttcagatc caaagaaggc gctgagccaa agcaaatcgg 4680 ccacttgggt gtcttgcatc ctgaagtcat gatgaatttc gacgttccat tcgctgcatc 4740 ctttgtagag gttaatgccg aagtcttcct ataatgtaat gttctaacaa aaatttttac 4800 tgatttataa aacttatata gatagataga catatataca tatctatata tagaaacaca 4860 actaaagttt accatgtttt atatagggca ccgagctcga attcactggc cgtcgtttta 4920 caacgtcgtg actgggaaaa ccgcggttac gtgcagtcga tgataagctg tcaaacatga 4980 gaattgtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt 5040 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg 5100 cggtetgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct 5160 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc 5220 ccagcaggcg aaaatcctgt Łtgatggtgg ttaacggcgg gatataacat gagctgtctt 5280 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa 5340 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga 5400 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt 5460 cccgttccgc tatcggctga atctgattgc gagtgagata cttatgccag ccagccagac 5520 gcagaegcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca 5580 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt 5640 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt 5700 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt 5760 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg 5820 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg 5880 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg 5940 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt 6000 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat 6060 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc 6120 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg caccattcga 6180 tggtgtcgga atttcgggca gcgttgggtc ctggccacgg gtgcgcatga cttaagcggt 6240 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct 6300 cgctcactga ctcgctgcgc tcggtctgtc ggctgcggcg agcggtatca gctcactcaa 6360 aggcggtaat acggttatce acagaatcag gggataacgc aggaaagaac atgtgagcaa 6420 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 6480 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 6540 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 6600 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 6660 ctcaatgctc acgctgtagg tateteagtt cggtgtaggt cgttcgctcc aagctgggct 6720 gtgtgcacga accccsegtt cagcccgacc getgegeett atccggtaac tatcgtcttg 6780 agtccaaccc ggtaagacac gaettatege cactggcagc agccactggt aacaggatta 6840 geagagegag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 6900

-103acactagaag gagttggtag gcaagcagca cggggtctga caaaaaggat gtatatatga cagegatctg cgatacggga caccggctcc gtcctgcaac gtagttcgcc cacgctcgtc catgatcccc gaagtaagtt ctgtcatgcc gagaatagtg cgccacatag tctcaaggat gatcttcagc atgccgcaaa ttcaatatta gtatttagaa acgtctaaga cctttcgtct &lt;210&gt; 3 &lt;211&gt; 1512 &lt;212&gt; DNA gacagtattt ctcttgatcc gattacgcgc cgctcagtgg cttcacctag gtaaacttgg tetatttcgt gggcttacca agatttatca tttatccgcc agttaatagt gtttggtatg catgttgtgc ggccgcagtg atccgtaaga tatgcggcga cagaacttta cttaccgctg atcttttact aaagggaata ttgaagcatt aaataaacaa aaccattatc tcac ggtatctgcg ggcaaacaaa agaaaaaaag aacgaaaacŁ atccttttaa tctgacagtt tcatecatag tctggcccca gcaataaacc tccatccagt ttgcgcaacg gcttcattca aaaaaagcgg ttatcactca tgcttttctg ccgagttgct aaagtgctca ttgagatcca ttcaccagcg agggcgacac tatcagggtt ataggggttc atcatgacat ctctgctgaa gccagttacc ttcggaaaaa 6960 ccaccgctgg tagcggtggt ttttttgttt 7020 gatctcaaga agatcccttg atcttttcta cacgttaagg 7080 gattttggtc atgagattat 7140 attaaaaatg aagttttaaa tcaatctaaa 7200 accaatgctt aatcagtgag gcacctatct 7260 ctgcctgact ccccgtegtg tagataacta 7320 gtgctgcaat gataccgcga gacccacgct 7330 agccagccgg aagggccgag cgcagaagtg 7440 ctattaattg ttgccgggaa gctagagtaa 7500 ttgttgecat Łgctacaggc atcgtggtgt 7560 gctccggttc ccaacgatca aggcgagtta 7620 ttagctcctt cggtcctccg atcgttgtca 7680 tggttatggc agcactgcat aattctctta 7740 tgactggtga gtactcaacc aagtcattct 7300 cttgcccggc gtcaatacgg gataataccg 7860 tcattggaaa acgttcttcg gggcgaaaac 7920 gttcgatgta acccactcgt gcacccaact 7980 tttctgggtg agcaaaaaca ggaaggcaaa 8040 ggaaacgttg aatactcata ctcttccttt 8100 attgtctcat gagcggatac atatttgaat 8160 cgcgcacatt tccccgaaaa gtgccacctg 8220 taacctataa aaatccggg

8294 &lt;213&gt; artificial sequence &lt;220&gt;

&lt;223&gt; alpha subunit of the yeast aminoacyl synthetase phenylalanine gene &lt;220&gt;

&lt;221&gt; miscfeature &lt;222&gt; 1234, 1235, 1236, 1243, 1244, 1245, 1252, 1253, 1254, 1309,

1310, 1311 &lt;223&gt; n = A, T, C or G &lt;400&gt; 3 atgtctgact tccaattaga aattctaaag aaactagatg aattggatga gatcaagtcc 60 acactggcaa ctttccctca gcacggctct caagatgttc tttccgcttt gaactctttg 120 aaagcccaca aćaagttaga gttttccaag gtcgacacgg ttacgtatga cttgaccaaa 190 gaaggtgctc aaattttgaa tgaaggttcg tacgaaatta aactagtcaa gctcatccaa 240 gagttgggtc aacttcaaat caaagatgtg atgtccaaac taggccctca agttggtaag 300 gtcggtcagg ctagagcttt caagaacggc tggatcgcca aaaacgcctc aaacgagctt 360 gaactctccg caaaattgca aaatacegat ttaaatgagc ttactgatga aacgcaatct 420 attctagcgc aaatcaagaa caactcgcat ctggatagca ttgacgccaa gattttgaac 480 gacttgaaga aaagaaagtt aattgctcaa ggtaaaatca cagatttcag tgtcaccaaa 540 gggccagagt tctcgaccga cctcaccaaa ttggaaaccg atcttacctc cgacatggtc 600 tccaccaatg catacaagga cttgaagttc aagccttaca atttcaattc tcaaggtgtg 660 caaatatctt caggtgctct tcacccctta aacaaagtca gagaggaatt tagacaaatt 720 ttcttttcca tgggattcac agagatgccc tcgaaccaat acgtcgagac aggtttctgg 700 aacttcgatg ccctttacgt cccacaacag catcctgctc gtgacctgca agacactttc 840 tacatcaagg acccactaac cgctgagttg cccgatgaca agacatacat ggacaatatc 900 aaagccgttc acgaacaggg gagattcggg tccatcggtt atcgttacaa ctggaagcca 960 gaagaatggcc 960 gaagaatggcc

-104cacgabbbgg ccaaagatcc aaagcccacc agattgtttt ctatcgaccg tgtbttccgt aacgaagcag ttgacgceac ccatttggce gaattccacc aggtggaagg tgttcttgcc gactacaaca ttactctggg tgacctgatc aagttcatgg aagagtttbt cgaaagaatg ggtgbcaccg gtttgagatt caagcctacc tacnnncctt acnnngagcc annnatggaa atcttttctt ggcacgaagg bbbgcaaaaa tgggtcgaaa tcggtaacnn nggtatgttc agaccagaaa tgctcgagtc catgggtcta ccaaaggatc taagagtcct bggbbggggg ttatccttgg aaagacctac cabgatcaaa tataaggttc aaaacatcag agaactgtta ggbcabaaag bctcbtbgga ctbbabcgaa accaatcctg cbgctagatb ggacgaagac ttgtacgaat aa &lt;210&gt; 4 &lt;211&gt; 13 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Artificial derivative of dihydrofolate reductase &lt;400&gt; 4

Asn Gly Asp Leu Pro Trp Pro Pro Leu Arg Asn Glu Lys 15 10 &lt;210&gt; 5 &lt;211&gt; 14 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

1080 114 0 1200 1260 1320 1380 1440 1500 1512 &lt;223&gt; Artificial derivative of dihydrofolate reductase &lt;220&gt;

&lt;221&gt; VARIANT &lt;222&gt; 13 &lt;223&gt; Xaa = suppressor amino acid &lt;400&gt; 5

Asn Gly Asp Leu Pro Trp Pro Pro Leu Arg Asn Glu Xaa Lys 15 10 &lt;210&gt; 6 &lt;211&gt; 8 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; An artificial derivative of dihydrofolate reductase

-105&lt;400&gt; 6

Pro Trp Pro Pro Leu Arg Aan Glu 1 S &lt;210&gt; 7 &lt;211&gt; 34 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Oligonucleotide primer &lt;400&gt; 7 cgattttcac acaggatcca gaccatgatt ctag 34 &lt;210&gt; 8 &lt;211&gt; 27 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; oligonucleotide primer &lt;400&gt; 8 gacggccagt gaattcgagc tcggtac 27 &lt;210&gt; 9 &lt;211&gt; 38 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Oligonucleotide primer &lt;400&gt; 9 ctacctacaa tccttacggc gagccatcaa tggaaatc &lt;210&gt; 10 &lt;211&gt; 38 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; oligonucleotide primer

-106 &lt;400&gt; 10 gatttccatt gatggctcgc cgtaaggatt gtaggtag 38 &lt;210&gt; 11 &lt;211&gt; 36 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Oligonucleotide primer &lt;400&gt; 11 ccgctcagga acgagtagaa gtacttccaa agaatg 36 &lt;210&gt; 12 &lt;211&gt; 36 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Oligonucleotide primer &lt;400&gt; 12 cattctttgg aagtacttct actcgttcct gagcgg 36 &lt;210&gt; 13 &lt;211&gt; 30 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Oligonucleotide primer &lt;400&gt; 13 gcactgacca tggctgaaca acacgcacag 30 &lt;210&gt; 14 &lt;211&gt; 31 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

-107 &lt;223&gt; Oligonucleotide primer &lt;400&gt; 14 ggacttcgga tcctttctgt gggcgcatcg c 31 &lt;210&gt; 15 &lt;211&gt; 14 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; alpha subunit of the yeast aminoacyl synthetase phenylalanine gene &lt;220&gt;

&lt;221&gt; VARIANT &lt;222&gt; 13 &lt;223&gt; Xaa = suppressor amino acid &lt;400&gt; 15

Asn Gly Asp Leu Pro Trp Pro Pro Leu Arg Asn Glu Xaa Lys 15 io &lt;210&gt; 16 &lt;211&gt; 39 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; oligonucleotide primer &lt;400&gt; 16 gaacacagga cctccacatt tagagtatgg cgctctccc 39 &lt;210&gt; 17 &lt;211&gt; 39 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; oligonucleotide primer &lt;400&gt; 17 gggagagcgc catactctaa atgtggaggt cctgtgttc 39 &lt;210&gt; 18

-108 &lt;211&gt; 42 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Oligonucleotide primer &lt;400&gt; 18 ctgggtaagc ttcgctaagg atctgccctg gtgcgaactc tg 42 &lt;210&gt; 19 &lt;211&gt; 43 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Oligonucleotide primer &lt;400&gt; 19 gattacggat tcctaatacg actcactata gcggacttag ctc 43 &lt;210&gt; 20 &lt;211&gt; 40 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Oligonucleotide primer &lt;400&gt; 20 cggaagcaga aagtgtaaag agcggggtgc ctaatgagtg 40 &lt;210&gt; 21 &lt;211&gt; 40 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; oligonucleotide primer &lt;400&gt; 21 cactcattag gcaccccgct ctttacactt tatgcttccg 40 &lt;210&gt; 22

-109 &lt;211&gt; 42 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Oligonucleotide primer &lt;400&gt; 22 cttgtcacta ctctgaccta tggtgttcaa tgcttctccc gt 42 &lt;210&gt; 23 &lt;211&gt; 42 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; oligonucleotide primer &lt;400&gt; 23 acgggagaag cattgaacac cataggtcag agtagtgaca ag &lt;210&gt; 24 &lt;211&gt; 39 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Oligonucleotide primer &lt;400&gt; 24 gtacaggaac gcactatatt cttcaaagat gacgggaac 39 &lt;210&gt; 25 &lt;211&gt; 39 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; oligonucleotide primer &lt;400&gt; 25 gttcccgtca tctttgaaga atatagtgcg ttcctgtac &lt;210&gt; 26

-110 &lt;211&gt; 39 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Oligonucleotide primer &lt;400&gt; 26 cacaatgtat acatcatggc agacaaacaa aagaatgga 39 &lt;210&gt; 27 &lt;211&gt; 39 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Oligonucleotide primer &lt;400&gt; 27 tccattcttt tgtttgtctg ccatgatgta tacattgtg 39 &lt;210&gt; 28 &lt;211&gt; 45 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Oligonucleotide primer &lt;400&gt; 28 gtgccacctg acgtctaaga aaccattatt atcatgacat taacc &lt;210&gt; 29 &lt;211&gt; 41 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; oligonucleotide primer &lt;400&gt; 29 gagtaaagga gaagaacttt ttactggagt tgtcccaatt c &lt;210&gt; 30

-111 &lt;211&gt; 41 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Oligonucleotide primer &lt;400&gt; 30 gaattgggac aactccagta aaaagttctt ctcctttact c 41 &lt;210&gt; 31 &lt;211&gt; 43 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Oligonucleotide primer &lt;400&gt; 31 ggccaacact tgtcactact tttacctatg gtgttcaatg ctt 43 &lt;210&gt; 32 &lt;211&gt; 43 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Oligonucleotide primer &lt;400&gt; 32 aagcattgaa caccataggt aaaagtagtg acaagtgttg gcc &lt;210&gt; 33 &lt;211&gt; 43 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; oligonucleotide primer &lt;400&gt; 33 catatgaaac ggcatgactt ttttaagagt gccatgcccg aag

-112 &lt;210&gt; 34 &lt;211&gt; 43 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Oligonucleotide primer &lt;400 34 cttcgggcat ggcactctta aaaaagtcat gccgtttcat atg 43 &lt;210&gt; 35 &lt;211&gt; 49 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Oligonucleotide primer &lt;400&gt; 35 gttatgtaca ggaacgcact atatttttca aagatgacgg gaactacaa 49 &lt;210&gt; 36 &lt;211&gt; 49 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Oligonucleotide primer &lt;400&gt; 36 ttgtagttcc cgtcatcttt gaaaaatata gtgcgttcct gtacataac 49 &lt;210&gt; 37 &lt;211&gt; 53 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; oligonucleotide primer &lt;400&gt; 37

-113acaaaagaat ggaatcaaag ctaactttaa aattcgccac aacattgaag atg &lt;210&gt; 38 &lt;211&gt; 53 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; oligonucleotide primer &lt;400&gt; 38 catcttcaat gttgtggcga attttaaagt tagctttgat tccattcttt tgt 53 &lt;210&gt; 39 &lt;211&gt; 39 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Oligonucleotide primer &lt;400&gt; 39 cgccaagcta gcttggattc tcaccaataa aaaacgccc 39 &lt;210&gt; 40 &lt;211&gt; 43 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; oligonucleotide primer &lt;400&gt; 40 tacctatggt gttcaatgct tttcccgtta tccggatcat atg 43 &lt;210&gt; 41 &lt;211&gt; 43 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; oligonucleotide primer

-114 &lt;400&gt; 41 catatgatcc ggataacggg aaaagcattg aacaccatag gta 43 &lt;210&gt; 42 &lt;211&gt; 50 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Oligonucleotide primer &lt;400&gt; 42 gttatgtaca ggaacgcact atatttttta aagatgacgg gaactacaag 50 &lt;210&gt; 43 &lt;211&gt; 50 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Oligonucleotide primer &lt;400&gt; 43 cttgtagttc ccgtcatctt taaaaaatat agtgcgttcc tgtacataac 50 &lt;210&gt; 44 &lt;211&gt; 43 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; oligonucleotide primer &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; 16, 17 &lt;223&gt; n = A, T, C or G &lt;400&gt; 44 gtcgaaatcg gtaacnnkgg tatgttcaga ccagaaatgc tcg &lt;210&gt; 45

-115 &lt;211&gt; 43 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; oligonucleotide primer &lt;220&gt;

&lt;221&gt; miscfeature &lt;222&gt; 27, 28 &lt;223&gt; n - A, T, C or G &lt;400&gt; 45 cgagcatttc tggtctgaac ataccmnngt taccgatttc gac 43 &lt;210&gt; 46 &lt;211&gt; 47 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt; 220&gt;

&lt;223&gt; oligonucleotide primer &lt;220&gt;

&lt;221&gt; miscfeature &lt;222&gt; 14, 15,23,24,32,33 &lt;223&gt; n = A, T, C or G &lt;400&gt; 46 caagcctacc tacnnkcctt acnnkgagcc annkatggaa atctttt &lt;210 47 &lt;211&gt; 47 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220 &lt;223&gt; Oligonucleotide primer &lt;220 &lt;221&gt; misc_feature &lt;222&gt; 15, 16, 24, 25,33,34

-116 &lt;223&gt; η = A, T, C or G &lt;400&gt; 47 aaaagatttc catmnntggc tcmnngtaag gmnngtaggt aggcttg 47 &lt;210&gt; 48 &lt;211&gt; 432 &lt;212&gt; PRT &lt;213&gt; Thermus thermophilus &lt;400&gt; 48

<td>Underworld 1</td><td>ala</td><td>Gly</td><td>Thr</td><td>Gly 5</td><td>His</td><td>Thr</td><td>Pro</td><td>Glu</td><td>Glu 10</td><td>ala</td><td>Leu</td><td>ala</td><td>Leu</td><td>Leu 15</td><td>lys</td>

<td>Arg</td><td>Gly</td><td>ala</td><td>Glu twenty</td><td>Glu</td><td>How much</td><td>val</td><td>Pro</td><td>Glu 25</td><td>Glu</td><td>Glu</td><td>Leu</td><td>Leu</td><td>ala thirty</td><td>lys</td><td>Leu</td>

<td>lys</td><td>Glu</td><td>Gly 35</td><td>Arg</td><td>Pro</td><td>Leu</td><td>Thr</td><td>val 40</td><td>lys</td><td>Leu</td><td>Gly</td><td>ala</td><td>Asp 45</td><td>Pro</td><td>Thr</td><td>Arg</td>

<td>Pro</td><td>Asp 50</td><td>Leu</td><td>His</td><td>Leu</td><td>Gly</td><td>His 55</td><td>ala</td><td>Val</td><td>val</td><td>Leu</td><td>Arg 60</td><td>lys</td><td>Underworld</td><td>Arg</td><td>Gin</td>

<td>phe 65</td><td>Gin</td><td>Glu</td><td>Leu</td><td>Gly</td><td>His 70</td><td>lys</td><td>val</td><td>val</td><td>Leu</td><td>How much 75</td><td>How much</td><td>Gly</td><td>Asp</td><td>phe</td><td>Thr 80</td>

<td>Gly</td><td>Underworld</td><td>how much</td><td>Gly</td><td>Asp 85</td><td>Pro</td><td>Cheese</td><td>Gly</td><td>Arg</td><td>Cheese 90</td><td>lys</td><td>Thr</td><td>Arg</td><td>Pro</td><td>Pro 95</td><td>Leu</td>

<td>Thr</td><td>Leu</td><td>Glu</td><td>Glu 100</td><td>Thr</td><td>Arg</td><td>Glu</td><td>own</td><td>ala 105</td><td>lys</td><td>Thr</td><td>Tyr</td><td>val</td><td>ala 110</td><td>Gin</td><td>ala</td>

<td>Gly</td><td>lys</td><td>How much 115</td><td>Leu</td><td>Arg</td><td>Gin</td><td>Glu</td><td>Pro 120</td><td>His</td><td>Leu</td><td>phe</td><td>Glu</td><td>Leu 12S</td><td>Arg</td><td>Tyr</td><td>own</td>

<td>Cheese</td><td>Glu 130</td><td>Trp</td><td>Leu</td><td>Glu</td><td>Gly</td><td>Leu 135</td><td>Thr</td><td>phe</td><td>lys</td><td>Glu</td><td>val 140</td><td>val</td><td>Arg</td><td>Leu</td><td>Thr</td>

<td>Cheese 14 5</td><td>Leu</td><td>Underworld</td><td>Thr</td><td>val</td><td>ala 150</td><td>Gin</td><td>Underworld</td><td>Leu</td><td>Glu</td><td>Arg 1S5</td><td>Glu</td><td>Asp</td><td>phe</td><td>lys</td><td>lys 160</td>

<td>Arg</td><td>Tyr</td><td>Glu</td><td>ala</td><td>Gly 165</td><td>How much</td><td>Pro</td><td>How much</td><td>Cheese</td><td>Leu 170</td><td>His</td><td>Glu</td><td>Leu</td><td>Leu</td><td>Tyr 175</td><td>Pro</td>

<td>phe</td><td>ala</td><td>Gin</td><td>ala 180</td><td>Tyr</td><td>Asp</td><td>Cheese</td><td>val</td><td>ala 1B5</td><td>How much</td><td>Arg</td><td>ala</td><td>Asp</td><td>val 190</td><td>Glu</td><td>Underworld</td>

<td>Gly</td><td>Gly</td><td>Thr 195</td><td>Asp</td><td>Gin</td><td>Arg</td><td>phe</td><td>own 200</td><td>Leu</td><td>Leu</td><td>val</td><td>Gly</td><td>Arg 205</td><td>Glu</td><td>val</td><td>Gin</td>

<td>Arg</td><td>ala 210</td><td>Tyr</td><td>Gly</td><td>Gin</td><td>Cheese</td><td>Pro 215</td><td>Gin</td><td>val</td><td>cys</td><td>phe</td><td>Leu 220</td><td>Underworld</td><td>Pro</td><td>Leu</td><td>Leu</td>

<td>val 225</td><td>Gly</td><td>Leu</td><td>Asp</td><td>Gly</td><td>Arg 230</td><td>Glu</td><td>lys</td><td>Underworld</td><td>Cheese</td><td>lys 235</td><td>Cheese</td><td>Leu</td><td>Asp</td><td>own</td><td>Tyr 240</td>

<td>How much</td><td>Gly</td><td>Leu</td><td>Thr</td><td>Glu 245</td><td>Pro</td><td>Pro</td><td>Glu</td><td>ala</td><td>Underworld 250</td><td>phe</td><td>lys</td><td>lys</td><td>Leu</td><td>Underworld 255</td><td>Arg</td>

<td>val</td><td>Pro</td><td>Asp</td><td>Pro 260</td><td>Leu</td><td>Leu</td><td>Pro</td><td>Cheese</td><td>Tyr 265</td><td>phe</td><td>Arg</td><td>Leu</td><td>Leu</td><td>Thr 270</td><td>Asp</td><td>Leu</td>

<td>Glu</td><td>Glu</td><td>Glu 275</td><td>Glu</td><td>How much</td><td>Glu</td><td>ala</td><td>Leu 280</td><td>Leu</td><td>lys</td><td>ala</td><td>Gly</td><td>Pro 285</td><td>val</td><td>Pro</td><td>ala</td>

<td>His</td><td>Arg</td><td>val</td><td>Leu</td><td>ala</td><td>Arg</td><td>Leu</td><td>Leu</td><td>Thr</td><td>ala</td><td>ala</td><td>Tyr</td><td>ala</td><td>Leu</td><td>Pro</td><td>Gin</td>

»»*&amp;*»

-117-

<td>How much</td><td>290 Pro</td><td>Pro</td><td>Arg</td><td>How much</td><td>Asp</td><td>295 Arg</td><td>ala</td><td>phe</td><td>Tyr</td><td>Glu</td><td>300 Cheese</td><td>Leu</td><td>Gly</td><td>Tyr</td><td>ala</td>

<td>305 Trp</td><td>Glu</td><td>ala</td><td>phe</td><td>Gly</td><td>310 Arg</td><td>Asp</td><td>lys</td><td>Glu</td><td>ala</td><td>315 Gly</td><td>Pro</td><td>Glu</td><td>Glu</td><td>val</td><td>320 Arg</td>

<td>Arg</td><td>ala</td><td>Glu</td><td>ala</td><td>325 Arg</td><td>Tyr</td><td>Asp</td><td>Glu</td><td>val</td><td>330 ala</td><td>lys</td><td>Gly</td><td>Gly</td><td>How much</td><td>335 Pro</td><td>Glu</td>

<td>Glu</td><td>How much</td><td>Pro</td><td>340 Glu</td><td>val</td><td>Thr</td><td>How much</td><td>Pro</td><td>345 ala</td><td>Cheese</td><td>Glu</td><td>Leu</td><td>lys</td><td>350 Glu</td><td>Gly</td><td>Arg</td>

<td>How much</td><td>Trp</td><td>355 val</td><td>ala</td><td>Arg</td><td>Leu</td><td>phe</td><td>360 Thr</td><td>Leu</td><td>ala</td><td>Gly</td><td>Leu</td><td>365 Thr</td><td>Pro</td><td>Cheese</td><td>own</td>

<td>ala</td><td>370 Glu</td><td>ala</td><td>Arg</td><td>Arg</td><td>Leu</td><td>375 How much</td><td>Gin</td><td>own</td><td>Arg</td><td>Gly</td><td>380 Leu</td><td>Arg</td><td>Leu</td><td>Asp</td><td>Gly</td>

<td>385 Glu</td><td>val</td><td>Leu</td><td>Thr</td><td>Asp</td><td>390 Pro</td><td>Underworld</td><td>Leu</td><td>Gin</td><td>val</td><td>395 Asp</td><td>Leu</td><td>Cheese</td><td>Arg</td><td>Pro</td><td>400 Arg</td>

<td>How much</td><td>Leu</td><td>Gin</td><td>Arg</td><td>405 Gly</td><td>lys</td><td>Asp</td><td>Arg</td><td>phe</td><td>410 val</td><td>Arg</td><td>val</td><td>Arg</td><td>Leu</td><td>415 Cheese</td><td>Asp</td>

<td></td><td></td><td></td><td> 420</td><td></td><td></td><td></td><td></td><td> 425</td><td></td><td></td><td></td><td></td><td> 430</td><td></td><td></td>

&lt;210&gt; 49 &lt;211&gt; 372 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 49

<td>Underworld 1</td><td>Gly</td><td>Asp</td><td>ala</td><td>Pro 5</td><td>Cheese</td><td>Pro</td><td>Glu</td><td>Glu</td><td>lys 10</td><td>Leu</td><td>His</td><td>Leu</td><td>How much</td><td>Thr 15</td><td>Arg</td>

<td>own</td><td>Leu</td><td>Gin</td><td>Glu twenty</td><td>val</td><td>Leu</td><td>Gly</td><td>Glu</td><td>□ lu 25</td><td>lys</td><td>Leu</td><td>lys</td><td>Glu</td><td>How much thirty</td><td>Leu</td><td>lys</td>

<td>Glu</td><td>Arg</td><td>Glu 35</td><td>Leu</td><td>lys</td><td>How much</td><td>Tyr</td><td>Trp 40</td><td>Gly</td><td>Thr</td><td>ala</td><td>Thr</td><td>Thr 45</td><td>Gly</td><td>Ly3</td><td>Pro</td>

<td>His</td><td>val 50</td><td>ala</td><td>Tyr</td><td>phe</td><td>val</td><td>Pro 55</td><td>Underworld</td><td>Cheese</td><td>lys</td><td>How much</td><td>ala 60</td><td>Asp</td><td>phe</td><td>Leu</td><td>lys</td>

<td>ala 65</td><td>Gly</td><td>Cys</td><td>Glu</td><td>val</td><td>Thr 70</td><td>How much</td><td>Leu</td><td>phe</td><td>ala</td><td>Asp 75</td><td>Leu</td><td>His</td><td>ala</td><td>Tyr</td><td>Leu 80</td>

<td>Asp</td><td>own</td><td>Underworld</td><td>lys</td><td>ala 85</td><td>Pro</td><td>Trp</td><td>Glu</td><td>Leu</td><td>Leu 90</td><td>Glu</td><td>Leu</td><td>Arg</td><td>val</td><td>Cheese 95</td><td>Tyr</td>

<td>.Tyr</td><td>Glu</td><td>own</td><td>val 100</td><td>How much</td><td>lys</td><td>ala</td><td>Underworld</td><td>Leu 105</td><td>Glu</td><td>Cheese</td><td>How much</td><td>Gly</td><td>val 110</td><td>Pro</td><td>Leu</td>

<td>Glu</td><td>lys</td><td>Leu 115</td><td>lys</td><td>phe</td><td>How much</td><td>lys</td><td>Gly 120</td><td>Thr</td><td>Asp</td><td>Tyr</td><td>Gin</td><td>Leu 125</td><td>Cheese</td><td>lys</td><td>Glu</td>

<td>Tyr</td><td>Thr 130</td><td>Leu</td><td>Asp</td><td>val</td><td>Tyr</td><td>Arg 13S</td><td>Leu</td><td>Cheese</td><td>Cheese</td><td>val</td><td>val 140</td><td>Thr</td><td>Gin</td><td>His</td><td>Asp</td>

<td>Cheese 145</td><td>lys</td><td>lys</td><td>ala</td><td>Gly</td><td>ala ISO</td><td>Glu</td><td>val</td><td>val</td><td>lys</td><td>Gin 155</td><td>val</td><td>Glu</td><td>His</td><td>Pro</td><td>Leu 160</td>

<td>.Leu</td><td>Cheese</td><td>Gly</td><td>Leu</td><td>Leu 165</td><td>Tyr</td><td>Pro</td><td>Gly</td><td>Leu</td><td>Gin 170</td><td>ala</td><td>Leu</td><td>Asp</td><td>Glu</td><td>Glu 175</td><td>Tyr</td>

<td>Leu</td><td>lys</td><td>val</td><td>Asp 180</td><td>ala</td><td>Gin</td><td>phe</td><td>Gly</td><td>Gly 185</td><td>How much</td><td>Asp</td><td>Gin</td><td>Arg</td><td>lys 190</td><td>How much</td><td>phe</td>

<td>Thr</td><td>phe</td><td>ala 195</td><td>Glu</td><td>lys</td><td>Tyr</td><td>Leu</td><td>Pro 200</td><td>ala</td><td>Leu</td><td>Gly</td><td>Tyr</td><td>Cheese 20S</td><td>lys</td><td>Arg</td><td>val</td>

<td>His</td><td>Leu 210</td><td>mec</td><td>own</td><td>Pro</td><td>mec</td><td>val 215</td><td>Pro</td><td>Gly</td><td>Leu</td><td>Thr</td><td>Gly 220</td><td>Cheese</td><td>lys</td><td>Underworld</td><td>Cheese</td>

<td>Cheese 225</td><td>Cheese</td><td>Glu</td><td>Glu</td><td>Glu</td><td>Cheese 230</td><td>lys</td><td>How much</td><td>ASp</td><td>Leu</td><td>Leu 235</td><td>Archbishop</td><td>Arg</td><td>lys</td><td>Glu</td><td>Asp 240</td>

<td>val</td><td>lys</td><td>lys</td><td>lys</td><td>Leu 245</td><td>lys</td><td>lys</td><td>ala</td><td>phe</td><td>Cys 250</td><td>Glu</td><td>Pro</td><td>Gly</td><td>own</td><td>val 255</td><td>Glu</td>

<td>own</td><td>own</td><td>Gly</td><td>val</td><td>Leu</td><td>Cheese</td><td>phe</td><td>How much</td><td>lys</td><td>His</td><td>val</td><td>Leu</td><td>phe</td><td>Pro</td><td>Leu</td><td>lys</td>

-118-

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>Cheese</td><td>Glu</td><td>phe</td><td>val</td><td>How much</td><td>Leu</td><td>Arg</td><td>Asp</td><td>Glu</td><td>lys</td><td>Trp</td><td>Gly</td><td>Gly</td><td>own</td><td>lys</td><td>Thr</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td>

<td>Tyr</td><td>Thr</td><td>ala</td><td>Tyr</td><td>val</td><td>Asp</td><td>Leu</td><td>Glu</td><td>lys</td><td>Asp</td><td>phe</td><td>ala</td><td>ala</td><td>Glu</td><td>val</td><td>val</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>His</td><td>Pro</td><td>Gly</td><td>Asp</td><td>Leu</td><td>lys</td><td>own</td><td>Cheese</td><td>val</td><td>Glu</td><td>val</td><td>ala</td><td>Leu</td><td>own</td><td>lys</td><td>Leu</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

<td>Leu</td><td>Asp</td><td>Pro</td><td>How much</td><td>Arg</td><td>Glu</td><td>lys</td><td>phe</td><td>own</td><td>Thr</td><td>Pro</td><td>ala</td><td>Leu</td><td>lys</td><td>lys</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td>

<td>ala</td><td>Cheese</td><td>ala</td><td>ala</td><td>Tyr</td><td>Pro</td><td>Asp</td><td>Pro</td><td>Cheese</td><td>Lye</td><td>Gin</td><td>lys</td><td>Pro</td><td>Underworld</td><td>ala</td><td>lys</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td>

<td>Gly</td><td>Pro</td><td>ala</td><td>lys</td><td>own</td><td>Cheese</td><td>Glu</td><td>Pro</td><td>Glu</td><td>Glu</td><td>val</td><td>How much</td><td>Leu</td><td>Glu</td><td>His</td><td>His</td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td><td></td><td></td><td> 355</td><td></td><td></td><td></td>

<td>His</td><td>His</td><td>His</td><td>His</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

370 &lt;210&gt; 50 &lt;211&gt; 424 &lt;212&gt; PRT &lt;213&gt; Escherichia coli &lt;400&gt; 50

<td>Underworld 1</td><td>ala</td><td>Cheese</td><td>Cheese</td><td>own 5</td><td>Leu</td><td>How much</td><td>lys</td><td>Gin</td><td>Leu 10</td><td>Gin</td><td>Glu</td><td>Arg</td><td>Gly</td><td>Leu 15</td><td>val</td>

<td>ala</td><td>Gin</td><td>val</td><td>Thr twenty</td><td>Asp</td><td>Glu</td><td>Glu</td><td>ala</td><td>Leu 25</td><td>ala</td><td>Glu</td><td>Arg</td><td>Leu</td><td>ala thirty</td><td>Gin</td><td>Gly</td>

<td>Pro</td><td>How much</td><td>ala 35</td><td>Leu</td><td>Tyr</td><td>Cys</td><td>Gly</td><td>phe 40</td><td>Asp</td><td>Pro</td><td>Thr</td><td>ala</td><td>Asp 45</td><td>Cheese</td><td>Leu</td><td>His</td>

<td>Leu</td><td>Gly 50</td><td>His</td><td>Leu</td><td>val</td><td>Pro</td><td>Leu 55</td><td>Leu</td><td>Cys</td><td>Leu</td><td>lys</td><td>Arg 60</td><td>phe</td><td>Gin</td><td>Gin</td><td>ala</td>

<td>Gly 65</td><td>His</td><td>lys</td><td>Pro</td><td>val</td><td>ala 70</td><td>Leu</td><td>val</td><td>Gly</td><td>Gly</td><td>ala 75</td><td>Thr</td><td>Gly</td><td>Leu</td><td>how much</td><td>Gly 80</td>

<td>Asp</td><td>Pro</td><td>Cheese</td><td>phe</td><td>lys 85</td><td>ala</td><td>ala</td><td>Glu</td><td>Arg</td><td>Ly3 90</td><td>Leu</td><td>own</td><td>Thr</td><td>Glu</td><td>Glu 95</td><td>Thr</td>

<td>val</td><td>Gin</td><td>Glu</td><td>Trp 100</td><td>val</td><td>Asp</td><td>lys</td><td>How much</td><td>Arg 105</td><td>lys</td><td>Gin</td><td>val</td><td>ala</td><td>Pro 110</td><td>phe</td><td>Leu</td>

<td>Asp</td><td>phe</td><td>Asp 115</td><td>cys</td><td>Gly</td><td>Glu</td><td>own</td><td>Cheese 120</td><td>ala</td><td>How much</td><td>ala</td><td>ala</td><td>own 125</td><td>own</td><td>Tyr</td><td>Asp</td>

<td>Trp</td><td>phe 130</td><td>Gly</td><td>own</td><td>Underworld</td><td>own</td><td>val 135</td><td>Leu</td><td>Thr</td><td>phe</td><td>Leu</td><td>Arg 140</td><td>Asp</td><td>How much</td><td>Gly</td><td>lys</td>

<td>His 145</td><td>phe</td><td>Cheese</td><td>val</td><td>own</td><td>Gin 150</td><td>Underworld</td><td>How much</td><td>own</td><td>lys</td><td>Glu 155</td><td>ala</td><td>val</td><td>lys</td><td>Gin</td><td>Arg 160</td>

<td>Leu</td><td>own</td><td>Arg</td><td>Glu</td><td>Asp 165</td><td>Gin</td><td>Gly</td><td>How much</td><td>Cheese</td><td>phe 170</td><td>Thr</td><td>Glu</td><td>phe</td><td>Cheese</td><td>Tyr 175</td><td>own</td>

<td>.Leu</td><td>Leu</td><td>Gin</td><td>Gly 160</td><td>Tyr</td><td>Asp</td><td>phe</td><td>ala</td><td>Cys 185</td><td>Leu</td><td>own</td><td>lys</td><td>Gin</td><td>Tyr 190</td><td>Gly</td><td>val</td>

<td>val</td><td>Leu</td><td>Gin 195</td><td>How much</td><td>Gly</td><td>Gly</td><td>Cheese</td><td>A3P 200</td><td>Gin</td><td>Trp</td><td>Gly</td><td>own</td><td>How much 205</td><td>Thr</td><td>Cheese</td><td>Gly</td>

<td>How much</td><td>Asp 210</td><td>Leu</td><td>Thr</td><td>Arg</td><td>Arg</td><td>Leu 215</td><td>His</td><td>Gin</td><td>own</td><td>Gin</td><td>val 220</td><td>phe</td><td>Gly</td><td>Leu</td><td>Thr</td>

<td>val 225</td><td>Pro</td><td>Leu</td><td>How much</td><td>Thr</td><td>lys 230</td><td>ala</td><td>Asp</td><td>Gly</td><td>Thr</td><td>lys 235</td><td>phe</td><td>Gly</td><td>lys</td><td>Thr</td><td>Glu 240</td>

<td>Gly</td><td>Gly</td><td>ala</td><td>val</td><td>Trp 245</td><td>Leu</td><td>Asp</td><td>Pro</td><td>lys</td><td>lys 250</td><td>Thr</td><td>Cheese</td><td>Pro</td><td>Tyr</td><td>lys 255</td><td>phe</td>

<td>Tyr</td><td>Gin</td><td>phe</td><td>Trp 260</td><td>How much</td><td>own</td><td>Thr</td><td>ala</td><td>Asp 265</td><td>ala</td><td>Asp</td><td>val</td><td>Tyr</td><td>Arg 270</td><td>phe</td><td>Leu</td>

<td>lys</td><td>phe</td><td>phe</td><td>Thr</td><td>phe</td><td>Underworld</td><td>Cheese</td><td>How much</td><td>Glu</td><td>Glu</td><td>How much</td><td>own</td><td>ala</td><td>Leu</td><td>Glu</td><td>Glu</td>

-119-

<td>Glu</td><td>Asp</td><td>275 lys</td><td>own</td><td>Cheese</td><td>Gly</td><td>lys</td><td>280 ala</td><td>Pro</td><td>Arg</td><td>ala</td><td>Gin</td><td>285 Tyr</td><td>val</td><td>Leu</td><td>ala</td>

<td>Glu</td><td>290 Gin</td><td>val</td><td>Thr</td><td>Arg</td><td>Leu</td><td>295 val</td><td>His</td><td>Gly</td><td>Glu</td><td>Glu</td><td>300 Gly</td><td>Leu</td><td>Gin</td><td>ala</td><td>ala</td>

<td>305 .Lys</td><td>Arg</td><td>How much</td><td>Thr</td><td>Glu</td><td>310 Cys</td><td>Leu</td><td>phe</td><td>Cheese</td><td>Gly</td><td>315 Cheese</td><td>Leu</td><td>Cheese</td><td>ala</td><td>Leu</td><td>320 Cheese</td>

<td>Glu</td><td>ala</td><td>Asp</td><td>phe</td><td>325 Glu</td><td>Gin</td><td>Leu</td><td>ala</td><td>Gin</td><td>330 Asp</td><td>Gly</td><td>val</td><td>Pro</td><td>Underworld</td><td>335 val</td><td>Glu</td>

<td>Underworld</td><td>Glu</td><td>lys</td><td>340 Gly</td><td>ala</td><td>Asp</td><td>Leu</td><td>Underworld</td><td>345 Gin</td><td>ala</td><td>Leu</td><td>val</td><td>Asp</td><td>350 Cheese</td><td>Glu</td><td>Leu</td>

<td>Gin</td><td>Pro</td><td>355 Cheese</td><td>Arg</td><td>Gly</td><td>Gin</td><td>ala</td><td>360 Arg</td><td>lys</td><td>Thr</td><td>How much</td><td>ala</td><td>365 Cheese</td><td>own</td><td>ala</td><td>How much</td>

<td>Thr</td><td>370 How much</td><td>own</td><td>Gly</td><td>Glu</td><td>lys</td><td>375 Gin</td><td>Cheese</td><td>Asp</td><td>Pro</td><td>Glu</td><td>380 Tyr</td><td>phe</td><td>phe</td><td>lys</td><td>Glu</td>

<td>385 Glu</td><td>Asp</td><td>Arg</td><td>Leu</td><td>phe</td><td>390 Gly</td><td>Arg</td><td>phe</td><td>Thr</td><td>Leu</td><td>395 Leu</td><td>Arg</td><td>Arg</td><td>Gly</td><td>lys</td><td>400 lys</td>

<td>own</td><td>Tyr</td><td>Cys</td><td>Leu 420</td><td>405 How much</td><td>Cys</td><td>Trp</td><td>lys</td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td>

&lt;210&gt; 51 &lt;211&gt; 442 &lt;212&gt; PRT &lt;213&gt; Drosophila melanogaster &lt;400&gt; 51

<td colspan="5">Met Cheese Gin Lys Asn</td><td colspan="3" rowspan="2">Leu Leu Glu</td><td colspan="7">Leu Thr Asp Arg Gly Phe Phe</td><td rowspan="2">His</td>

<td colspan="2"> 1</td><td colspan="3">s</td><td colspan="6"> 10</td><td> 15</td>

<td>Gly</td><td>How much</td><td>phe</td><td>Pro</td><td>Asp</td><td>Thr</td><td>ala</td><td>ala</td><td>Pro</td><td>Arg</td><td>Underworld</td><td>lys</td><td>Gin</td><td>Leu</td><td>phe</td><td>Thr</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Arg</td><td>Gly</td><td>Gin</td><td>Gin</td><td>Cheese</td><td>How much</td><td>Tyr</td><td>ala</td><td>Gly</td><td>phe</td><td>Asp</td><td>Pro</td><td>Thr</td><td>ala</td><td>Asp</td><td>Cheese</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Leu</td><td>His</td><td>val</td><td>Gly</td><td>own</td><td>Leu</td><td>Leu</td><td>val</td><td>How much</td><td>Underworld</td><td>Gly</td><td>Leu</td><td>How much</td><td>His</td><td>Cya</td><td>Gin</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Arg</td><td>ala</td><td>Gly</td><td>His</td><td>Arg</td><td>Pro</td><td>How much</td><td>ala</td><td>Leu</td><td>val</td><td>Gly</td><td>Gly</td><td>ala</td><td>Thr</td><td>Gly</td><td>Leu</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>How much</td><td>Gly</td><td>Asp</td><td>Pro</td><td>Cheese</td><td>Gly</td><td>Arg</td><td>lys</td><td>Thr</td><td>Glu</td><td>Arg</td><td>own</td><td>Gin</td><td>Leu</td><td>Gly</td><td>Glu</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Thr</td><td>val</td><td>How much</td><td>Glu</td><td>Thr</td><td>own</td><td>Leu</td><td>lys</td><td>ala</td><td>How much</td><td>Glu</td><td>Gin</td><td>Gin</td><td>Leu</td><td>Arg</td><td>Arg</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>val</td><td>phe</td><td>Glu</td><td>own</td><td>His</td><td>Glu</td><td>own</td><td>Cys</td><td>Leu</td><td>Trp</td><td>Asp</td><td>Cheese</td><td>lys</td><td>lys</td><td>Gin</td><td>lys</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Leu</td><td>Pro</td><td>Leu</td><td>ala</td><td>Pro</td><td>Leu</td><td>How much</td><td>how much</td><td>val</td><td>own</td><td>own</td><td>ala</td><td>Asp</td><td>Trp</td><td>Tyr</td><td>ala</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Asp</td><td>Leu</td><td>Gin</td><td>Leu</td><td>How much</td><td>Asp</td><td>phe</td><td>val</td><td>ala</td><td>own</td><td>Underworld</td><td>Gly</td><td>Arg</td><td>His</td><td>phe</td><td>Arg</td>

<td>14S</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Underworld</td><td>Gly</td><td>Cheese</td><td>Underworld</td><td>Leu</td><td>Cheese</td><td>Arg</td><td>Cheese</td><td>Cheese</td><td>val</td><td>Gin</td><td>Cheese</td><td>Arg</td><td>Leu</td><td>Glu</td><td>Cheese</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Glu</td><td>Asp</td><td>Gly</td><td>Underworld</td><td>Cheese</td><td>phe</td><td>Thr</td><td>Glu</td><td>phe</td><td>Thr</td><td>Tyr</td><td>Gin</td><td>How much</td><td>phe</td><td>Gin</td><td>ala</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Tyr</td><td>ASp</td><td>Trp</td><td>Leu</td><td>His</td><td>Leu</td><td>Leu</td><td>Arg</td><td>Arg</td><td>His</td><td>own</td><td>Cys</td><td>CYS</td><td>phe</td><td>Gin</td><td>Underworld</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Gly</td><td>Gly</td><td>Cheese</td><td>Asp</td><td>Gin</td><td>Thr</td><td>Gly</td><td>own</td><td>Leu</td><td>Underworld</td><td>Thr</td><td>Gly</td><td>His</td><td>Glu</td><td>Leu</td><td>How much</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Cheese</td><td>Arg</td><td>val</td><td>Glu</td><td>Arg</td><td>Lye</td><td>Arg</td><td>Glu</td><td>val</td><td>phe</td><td>Gly</td><td>Leu</td><td>Thr</td><td>Leu</td><td>Pro</td><td>Leu</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>val</td><td>Thr</td><td>Thr</td><td>Glu</td><td>Glu</td><td>Gly</td><td>Asp</td><td>lys</td><td>phe</td><td>Gly</td><td>lys</td><td>Cheese</td><td>ala</td><td>Gly</td><td>own</td><td>ala</td>

-120-

<td>val</td><td>Trp</td><td>Leu</td><td>ASp</td><td>245 Gly</td><td>own</td><td>lys</td><td>Thr</td><td>Cheese</td><td>2S0 Pro</td><td>phe</td><td>ala</td><td>Leu</td><td>Tyr</td><td>255 Gin</td><td>phe</td>

<td>phe</td><td>Leu</td><td>Arg</td><td>260 mec</td><td>Pro</td><td>Asp</td><td>cheese</td><td>Glu</td><td>265 val</td><td>Glu</td><td>Ly3</td><td>Leu</td><td>Leu</td><td>270 lys</td><td>Leu</td><td>phe</td>

<td>Thr</td><td>phe</td><td>275 How much</td><td>Pro</td><td>Leu</td><td>Pro</td><td>Gin</td><td>280 val</td><td>Glu</td><td>Gin</td><td>Leu</td><td>Underworld</td><td>285 Arg</td><td>Glu</td><td>Kis</td><td>Thr</td>

<td>lys</td><td>290 Glu</td><td>Pro</td><td>Glu</td><td>lys</td><td>Arg</td><td>295 lys</td><td>ala</td><td>Gin</td><td>Thr</td><td>Leu</td><td>300 Leu</td><td>ala</td><td>Glu</td><td>Asp</td><td>val</td>

<td>305 Thr</td><td>Leu</td><td>Leu</td><td>val</td><td>His</td><td>310 Gly</td><td>Glu</td><td>Cheese</td><td>Gly</td><td>Leu</td><td>315 lys</td><td>Gin</td><td>ala</td><td>Glu</td><td>Arg</td><td>320 val</td>

<td>Thr</td><td>own</td><td>ala</td><td>Leu</td><td>325 Tyr</td><td>lys</td><td>Gly</td><td>own</td><td>val</td><td>330 Glu</td><td>Gly</td><td>Leu</td><td>ala</td><td>Glu</td><td>335 Leu</td><td>own</td>

<td>Leu</td><td>Cheese</td><td>Glu</td><td>340 How much</td><td>Gin</td><td>Gin</td><td>Thr</td><td>phe</td><td>345 Gin</td><td>Gly</td><td>ala</td><td>Thr</td><td>Underworld</td><td>350 val</td><td>own</td><td>Leu</td>

<td>Leu</td><td>Thr</td><td>355 Glu</td><td>Pro</td><td>Gly</td><td>Underworld</td><td>Cheese</td><td>360 How much</td><td>Leu</td><td>Glu</td><td>Leu</td><td>ala</td><td>365 Underworld</td><td>lys</td><td>ala</td><td>lys</td>

<td>Cys</td><td>370 phe</td><td>Pro</td><td>Thr</td><td>Glu</td><td>Thr</td><td>375 Asp</td><td>ala</td><td>val</td><td>Arg</td><td>How much</td><td>380 How much</td><td>own</td><td>ala</td><td>Gly</td><td>Gly</td>

<td>385 phe</td><td>Tyr</td><td>val</td><td>own</td><td>Gin</td><td>390 lys</td><td>Arg</td><td>val</td><td>Gin</td><td>own</td><td>395 How much</td><td>ala</td><td>Glu</td><td>val</td><td>Leu</td><td>400 Thr</td>

<td>Thr</td><td>Gly</td><td>val</td><td>His</td><td>405 how much</td><td>Leu</td><td>Arg</td><td>own</td><td>Gly</td><td>410 How much</td><td>Cheese</td><td>Leu</td><td>Leu</td><td>Arg</td><td>415 val</td><td>Gly</td>

<td>lys</td><td>Arg</td><td>own 435</td><td>420 phe</td><td>Tyr</td><td>how much</td><td>val</td><td>Arg 440</td><td>425 Trp</td><td>Gin</td><td></td><td></td><td></td><td> 430</td><td></td><td></td>

&lt;210&gt; 52 &lt;211&gt; 492 &lt;212&gt; PRT &lt;213&gt; Saccharomyces cerevisiae &lt;400&gt; 52

<td>Underworld 1</td><td>Leu</td><td>Glu</td><td>Leu</td><td>Arg 5</td><td>Cheese</td><td>Cys</td><td>Cheese</td><td>own</td><td>Leu 10</td><td>val</td><td>own</td><td>Cheese</td><td>Cheese</td><td>Arg 15</td><td>Arg</td>

<td>Leu</td><td>val</td><td>pro</td><td>Leu twenty</td><td>val</td><td>Thr</td><td>Tyr</td><td>Cheese</td><td>Gly 25</td><td>Leu</td><td>Cheese</td><td>ala</td><td>How much</td><td>Thr thirty</td><td>Leu</td><td>Pro</td>

<td>lys</td><td>Cheese</td><td>Arg 15</td><td>phe</td><td>Tyr</td><td>Cheese</td><td>Gin</td><td>Pro 40</td><td>Cheese</td><td>ala</td><td>Leu</td><td>Glu</td><td>val 45</td><td>Gin</td><td>Gly</td><td>Thr</td>

<td>Cheese</td><td>Asp 50</td><td>Cheese</td><td>Arg</td><td>Cheese</td><td>Asp</td><td>own 55</td><td>How much</td><td>Leu</td><td>Asp</td><td>Glu</td><td>Leu 60</td><td>lys</td><td>Gin</td><td>Arg</td><td>Gly</td>

<td>Leu 65</td><td>val</td><td>Cheese</td><td>Gin</td><td>val</td><td>Cheese 70</td><td>Gin</td><td>Pro</td><td>Glu</td><td>Cheese</td><td>phe 75</td><td>Leu</td><td>Arg</td><td>Thr</td><td>lys</td><td>Leu 80</td>

<td>own</td><td>Gly</td><td>own</td><td>Asp</td><td>lys 85</td><td>How much</td><td>lys</td><td>Leu</td><td>Tyr</td><td>Cys 90</td><td>Gly</td><td>val</td><td>Asp</td><td>Pro</td><td>Thr 95</td><td>ala</td>

<td>Gin</td><td>Cheese</td><td>Leu</td><td>His 100</td><td>Leu</td><td>Gly</td><td>own</td><td>Leu</td><td>val 105</td><td>Pro</td><td>Leu</td><td>Underworld</td><td>val</td><td>Leu 110</td><td>Leu</td><td>His</td>

<td>phe</td><td>Tyr</td><td>val 115</td><td>lys</td><td>Gly</td><td>His</td><td>Αβρ</td><td>How much 120</td><td>val</td><td>Thr</td><td>val</td><td>how much</td><td>Gly 125</td><td>Gly</td><td>ala</td><td>Thr</td>

<td>Gly</td><td>Ly9 130</td><td>val</td><td>Gly</td><td>Asp</td><td>Pro</td><td>Cheese 135</td><td>Gly</td><td>Arg</td><td>lys</td><td>Thr</td><td>Glu 140</td><td>Arg</td><td>Asp</td><td>val</td><td>Underworld</td>

<td>Glu 145</td><td>own</td><td>Asp</td><td>How much</td><td>Arg</td><td>Gin 150</td><td>Cheese</td><td>own</td><td>val</td><td>ala</td><td>Cheese 155</td><td>How much</td><td>Cheese</td><td>Gin</td><td>Gin</td><td>Leu 160</td>

<td>Gin</td><td>Arg</td><td>phe</td><td>phe</td><td>lys 165</td><td>own</td><td>Gly</td><td>Leu</td><td>Glu</td><td>Tyr 170</td><td>Tyr</td><td>Arg</td><td>own</td><td>Arg</td><td>Cys 175</td><td>ala</td>

<td>Leu</td><td>Thr</td><td>Glu</td><td>Asp 180</td><td>val</td><td>prp</td><td>Cheese</td><td>Gly</td><td>lys 185</td><td>Tyr</td><td>Thr</td><td>Pro</td><td>Arg</td><td>own 190</td><td>own</td><td>phe</td>

<td>aan</td><td>Trp</td><td>Trp</td><td>lys</td><td>Asp</td><td>How much</td><td>lys</td><td>Underworld</td><td>Leu</td><td>Asp</td><td>phe</td><td>Leu</td><td>ala</td><td>Asp</td><td>phe</td><td>Gly</td>

-121-

<td>Arg</td><td>His</td><td>195 how much</td><td>Arg</td><td>val</td><td>Gin</td><td>Cheese</td><td>200 Underworld</td><td>Leu</td><td>ala</td><td>Arg</td><td>Asp</td><td>205 cheese</td><td>How much</td><td>Cheese</td><td>Cheese</td>

<td>Arg</td><td>210 Leu</td><td>Gin</td><td>Thr</td><td>lys</td><td>own</td><td>215 Gly</td><td>Leu</td><td>Gly</td><td>phe</td><td>own</td><td>220 Glu</td><td>phe</td><td>Thr</td><td>Tyr</td><td>Gin</td>

<td>225 val</td><td>Leu</td><td>Gin</td><td>ala</td><td>Tyr</td><td>230 Asp</td><td>phe</td><td>Tyr</td><td>His</td><td>Leu</td><td>235 Tyr</td><td>lys</td><td>Glu</td><td>Glu</td><td>own</td><td>240 val</td>

<td>Thr</td><td>How much</td><td>Gin</td><td>val</td><td>245 Gly</td><td>Gly</td><td>own</td><td>Asp</td><td>Gin</td><td>250 Trp</td><td>Gly</td><td>own</td><td>How much</td><td>Thr</td><td>255 ala</td><td>Gly</td>

<td>How much</td><td>Asp</td><td>Leu</td><td>260 How much</td><td>own</td><td>Arg</td><td>How much</td><td>Gin</td><td>265 Pro</td><td>how much</td><td>lys</td><td>own</td><td>lys</td><td>270 Gly</td><td>Leu</td><td>Pro</td>

<td>phe</td><td>Gly</td><td>275 how much</td><td>Thr</td><td>val</td><td>Pro</td><td>Leu</td><td>280 Leu</td><td>Thr</td><td>Thr</td><td>ala</td><td>Thr</td><td>285 Gly</td><td>Glu</td><td>lys</td><td>phe</td>

<td>Gly</td><td>290 lys</td><td>Cheese</td><td>ala</td><td>Gly</td><td>own</td><td>295 ala</td><td>val</td><td>phe</td><td>How much</td><td>Asp</td><td>300 Pro</td><td>Cheese</td><td>How much</td><td>own</td><td>Thr</td>

<td>305 ala</td><td>Tyr</td><td>Asp</td><td>val</td><td>Tyr</td><td>310 Gin</td><td>phe</td><td>phe</td><td>Tyr</td><td>own</td><td>315 Thr</td><td>Leu</td><td>Asp</td><td>ala</td><td>Asp</td><td>320 val</td>

<td>Pro</td><td>lys</td><td>phe</td><td>Leu</td><td>32S lys</td><td>how much</td><td>phe</td><td>Thr</td><td>phe</td><td>330 Leu</td><td>own</td><td>Cheese</td><td>cheese</td><td>Glu</td><td>335 How much</td><td>lys</td>

<td>lys</td><td>How much</td><td>val</td><td>340 Glu</td><td>Thr</td><td>His</td><td>How much</td><td>lys</td><td>345 Cheese</td><td>Pro</td><td>Cheese</td><td>Leu</td><td>Arg</td><td>350 Tyr</td><td>Gly</td><td>Gin</td>

<td>Thr</td><td>Leu</td><td>355 Leu</td><td>ala</td><td>lys</td><td>Glu</td><td>val</td><td>360 Thr</td><td>Asp</td><td>Underworld</td><td>Leu</td><td>Tyr</td><td>365 Gly</td><td>val</td><td>Gly</td><td>Cheese</td>

<td>Gly</td><td>370 Cheese</td><td>Asp</td><td>Cheese</td><td>Glu</td><td>ala</td><td>375 Leu</td><td>Cheese</td><td>own</td><td>He</td><td>How much</td><td>380 phe</td><td>Gly</td><td>Arg</td><td>Tyr</td><td>Asp</td>

<td>385 Gly</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>ala</td><td>390 ala</td><td>lys</td><td>Leu</td><td>val</td><td>Asp</td><td>39S Leu</td><td>Cye</td><td>Lye</td><td>lys</td><td>ala</td><td>400 Arg</td>

<td>How much</td><td>Leu</td><td>Gin</td><td>Tyr</td><td>405 ala</td><td>Asp</td><td>Arg</td><td>Glu</td><td>How much</td><td>410 Asp</td><td>Leu</td><td>How much</td><td>lys</td><td>Leu</td><td>415 How much</td><td>Cys</td>

<td>lys</td><td>Leu</td><td>val</td><td>420 own</td><td>Cys</td><td>Cheese</td><td>val</td><td>Cheese</td><td>425 Glu</td><td>ala</td><td>Arg</td><td>Arg</td><td>lys</td><td>430 Leu</td><td>Cheese</td><td>Gin</td>

<td>Gly</td><td>Cheese</td><td>435 val</td><td>Tyr</td><td>Leu</td><td>His</td><td>His</td><td>440 Cheese</td><td>lys</td><td>Cheese</td><td>lys</td><td>val</td><td>445 own</td><td>Glu</td><td>own</td><td>How much</td>

<td>Cheese</td><td>450 own</td><td>Leu</td><td>ala</td><td>Pro</td><td>phe</td><td>455 Leu</td><td>How much</td><td>ASP</td><td>Asp</td><td>Arg</td><td>460 VAL</td><td>Leu</td><td>How much</td><td>Leu</td><td>Arg</td>

<td>465 How much</td><td>Gly</td><td>lys</td><td>Gin</td><td>lys 485</td><td>470 Cys</td><td>phe</td><td>How much</td><td>How much</td><td>Glu 4 90</td><td>475 Underworld</td><td>Arg</td><td></td><td></td><td></td><td> 480</td>

&lt;210&gt; 53 &lt;211&gt; 445 &lt;212&gt; PRT &lt;213&gt; Schizosaccharomyces pombe &lt;400 53

<td rowspan="2">Underworld 1</td><td colspan="4" rowspan="2">Arg Leu Leu cheese 5</td><td colspan="2" rowspan="2">Ala Cys</td><td rowspan="2">Leu</td><td rowspan="2">Lye</td><td colspan="7">Gin Leu Gin Ala Arg Ser Leu</td>

<td colspan="2"> 10</td><td colspan="5"> 15</td>

<td>How much</td><td>His</td><td>own</td><td>Thr</td><td>Thr</td><td>Leu</td><td>Leu</td><td>Gin</td><td>Pro</td><td>Cheese</td><td>Cys</td><td>own</td><td>val</td><td>own</td><td>Cheese</td><td>val</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Tyr</td><td>Leu</td><td>Gly</td><td>ala</td><td>Asp</td><td>Pro</td><td>Thr</td><td>ala</td><td>ala</td><td>Cheese</td><td>Leu</td><td>His</td><td>val</td><td>Gly</td><td>own</td><td>Leu</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>val</td><td>ala</td><td>Leu</td><td>checkmate</td><td>Pro</td><td>Leu</td><td>val</td><td>His</td><td>phe</td><td>phe</td><td>Leu</td><td>own</td><td>Gly</td><td>phe</td><td>Pro</td><td>val</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>phe</td><td>Thr</td><td>val</td><td>how much</td><td>Gly</td><td>Asp</td><td>ala</td><td>Thr</td><td>ala</td><td>Gin</td><td>Leu</td><td>Gly</td><td>ASp</td><td>pro</td><td>Cheese</td><td>Gly</td>

<td> 65</td><td></td><td></td><td></td><td></td><td>⁷⁰</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Arg</td><td>Cheese</td><td>Thr</td><td>Cheese</td><td>Arg</td><td>lys</td><td>Gin</td><td>Underworld</td><td>ala</td><td>Glu</td><td>Thr</td><td>Thr</td><td>Arg</td><td>Thr</td><td>ala</td><td>own</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Cheese</td><td>own</td><td>Cheese</td><td>how much</td><td>His</td><td>own</td><td>Gin</td><td>Leu</td><td>lys</td><td>Asp</td><td>Leu</td><td>Cheese</td><td>Cheese</td><td>Cheese</td><td>How much</td><td>Leu</td>

-122-

<td>Cheese</td><td>Tyr</td><td>ala</td><td>100 Gln</td><td>Asp</td><td>Cys</td><td>own</td><td>Tyr</td><td>105 Pro</td><td>phe</td><td>Cheese</td><td>Gln</td><td>Underworld</td><td>110 Pro</td><td>Cheese</td><td>Cheese</td>

<td>Cheese</td><td>Gln</td><td>115 Trp</td><td>Cheese</td><td>How much</td><td>val</td><td>Arg</td><td>120 own</td><td>Cheese</td><td>Cheese</td><td>Trp</td><td>Tyr</td><td>125 Glu</td><td>own</td><td>Leu</td><td>lys</td>

<td>Leu</td><td>130 Leu</td><td>lys</td><td>phe</td><td>Leu</td><td>Cheese</td><td>135 Cheese</td><td>val</td><td>Gly</td><td>Pro</td><td>His</td><td>140 val</td><td>Arg</td><td>val</td><td>Cheese</td><td>Gln</td>

<td>145 Underworld</td><td>Leu</td><td>ala</td><td>Arg</td><td>Asp</td><td>150 Cheese</td><td>val</td><td>Thr</td><td>Thr</td><td>Arg</td><td>155 Leu</td><td>Gln</td><td>Cheese</td><td>Pro</td><td>Cheese</td><td>160 Gly</td>

<td>Leu</td><td>Cheese</td><td>phe</td><td>ala</td><td>165 Glu</td><td>Leu</td><td>Thr</td><td>Tyr</td><td>Gln</td><td>170 Leu</td><td>Leu</td><td>Gln</td><td>ala</td><td>Tyr</td><td>175 Asp</td><td>Tyr</td>

<td>Cheese</td><td>Tyr</td><td>Leu</td><td>180 Tyr</td><td>Glu</td><td>own</td><td>His</td><td>Cheese</td><td>185 val</td><td>own</td><td>Leu</td><td>Gln</td><td>How much</td><td>190 Gly</td><td>Gly</td><td>Cheese</td>

<td>Asp</td><td>Gln</td><td>195 Trp</td><td>Gly</td><td>own</td><td>How much</td><td>Thr</td><td>200 ala</td><td>Gly</td><td>Thr</td><td>Asp</td><td>Leu</td><td>205 val</td><td>Arg</td><td>Arg</td><td>Thr</td>

<td>His</td><td>210 Pro</td><td>own</td><td>ala</td><td>own</td><td>val</td><td>215 Tyr</td><td>ala</td><td>Leu</td><td>Thr</td><td>Thr</td><td>220 Pro</td><td>Leu</td><td>Leu</td><td>Thr</td><td>Cheese</td>

<td>225 Cheese</td><td>Cheese</td><td>Gly</td><td>Gln</td><td>lys</td><td>230 Leu</td><td>Gly</td><td>lys</td><td>Cheese</td><td>ala</td><td>23S Gly</td><td>own</td><td>ala</td><td>how much</td><td>Trp</td><td>240 Leu</td>

<td>Asp</td><td>Pro</td><td>lys</td><td>Leu</td><td>245 Thr</td><td>Asp</td><td>Cheese</td><td>Tyr</td><td>Cheese</td><td>250 Leu</td><td>Tyr</td><td>Gln</td><td>Tyr</td><td>phe</td><td>255 how much</td><td>Cheese</td>

<td>ala</td><td>Pro</td><td>Asp</td><td>280 Asp</td><td>Leu</td><td>ala</td><td>Cys</td><td>lys</td><td>265 Cys</td><td>Leu</td><td>Asp</td><td>Underworld</td><td>Leu</td><td>270 Thr</td><td>Leu</td><td>Leu</td>

<td>Pro</td><td>Leu</td><td>275 Glu</td><td>Gln</td><td>Leu</td><td>Glu</td><td>Gln</td><td>280 How much</td><td>lys</td><td>ala</td><td>Glu</td><td>His</td><td>285 Glu</td><td>lys</td><td>Asp</td><td>Pro</td>

<td>cheese</td><td>290 Gln</td><td>Arg</td><td>how much</td><td>val</td><td>His</td><td>29S lys</td><td>Tyr</td><td>Leu</td><td>ala</td><td>Cheese</td><td>300 own</td><td>val</td><td>val</td><td>Arg</td><td>Underworld</td>

<td>305 val</td><td>His</td><td>Gly</td><td>lys</td><td>lys</td><td>310 ala</td><td>Leu</td><td>Glu</td><td>Leu</td><td>ala</td><td>315 Gln</td><td>How much</td><td>Gln</td><td>Thr</td><td>lys</td><td>320 Leu</td>

<td>Leu</td><td>His</td><td>Gly</td><td>ala</td><td>325 His</td><td>Gln</td><td>ala</td><td>Pro</td><td>phe</td><td>330 Gly</td><td>phe</td><td>Tyr</td><td>Cheese</td><td>Glu</td><td>335 ala</td><td>Pro</td>

<td>Gln</td><td>Gln</td><td>Gly</td><td>340 Asp</td><td>Cheese</td><td>phe</td><td>Pro</td><td>Cheese</td><td>345 Leu</td><td>Pro</td><td>Glu</td><td>How much</td><td>Arg</td><td>350 ala</td><td>Leu</td><td>phe</td>

<td>lys</td><td>Asp</td><td>355 Cys</td><td>lys</td><td>phe</td><td>Tyr</td><td>Arg</td><td>360 Thr</td><td>How much</td><td>Asp</td><td>Cheese</td><td>Cheese</td><td>365 How much</td><td>lys</td><td>Asp</td><td>Gln</td>

<td>Pro</td><td>370 phe</td><td>Cheese</td><td>Arg</td><td>Leu</td><td>Leu</td><td>375 Arg</td><td>Thr</td><td>Leu</td><td>Gln</td><td>How much</td><td>380 Tyr</td><td>Thr</td><td>Cheese</td><td>Arg</td><td>lys</td>

<td>385 Glu</td><td>ala</td><td>Thr</td><td>Glu</td><td>His</td><td>390 How much</td><td>Leu</td><td>Cheese</td><td>Gly</td><td>ala</td><td>395 val</td><td>Cheese</td><td>Leu</td><td>Gly</td><td>His</td><td>400 lys</td>

<td>Pro</td><td>How much</td><td>Leu</td><td>Asp</td><td>405 Cheese</td><td>own</td><td>Tyr</td><td>lys</td><td>phe</td><td>410 Pro</td><td>Asp</td><td>own</td><td>Cheese</td><td>Leu</td><td>415 phe</td><td>val</td>

<td>Leu</td><td>Arg</td><td>ala 435</td><td>420 Gly</td><td>lys</td><td>Arg</td><td>Thr</td><td>phe 440</td><td>42S val</td><td>Leu</td><td>Asp</td><td>Cheese</td><td>Leu 445</td><td> 430</td><td></td><td></td>

&lt;210&gt; 54 &lt;211&gt; 528 &lt;212&gt; PRT &lt;213&gt; Xenopus laevis &lt;400&gt; 54

<td>Underworld</td><td>Gly</td><td>Asp</td><td>Cheese</td><td>Leu</td><td>Thr</td><td>Leu</td><td>Glu</td><td>Gly</td><td>lys</td><td>ala</td><td>Gln</td><td>Leu</td><td>How much</td><td>Thr</td><td>Arg</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>own</td><td>Leu</td><td>Gln</td><td>Glu</td><td>Leu</td><td>Leu</td><td>Gly</td><td>Glu</td><td>Asp</td><td>lys</td><td>Underworld</td><td>lys</td><td>Glu</td><td>How much</td><td>Leu</td><td>lys</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Glu</td><td>Arg.</td><td>Pro</td><td>Leu</td><td>Arg</td><td>How much</td><td>Tyr</td><td>Trp</td><td>Gly</td><td>Thr</td><td>ala</td><td>Thr</td><td>Thr</td><td>Gly</td><td>lys</td><td>Pro</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>His</td><td>val</td><td>ala</td><td>Tyr</td><td>phe</td><td>val</td><td>Pro</td><td>Underworld</td><td>cheese</td><td>lys</td><td>How much</td><td>ala</td><td>Asp</td><td>phe</td><td>Leu</td><td>lys</td>

-123-

<td>ala</td><td>50 Gly</td><td>Cys</td><td>Glu</td><td>val</td><td>Thr</td><td>55 How much</td><td>Leu</td><td>phe</td><td>ala</td><td>Asp</td><td>60 Leu</td><td>His</td><td>ala</td><td>Tyr</td><td>Leu</td>

<td>«5 Asp</td><td>own</td><td>Underworld</td><td>lys</td><td>ala</td><td>70 Pro</td><td>Trp</td><td>Asp</td><td>Leu</td><td>Leu</td><td>75 Glu</td><td>Leu</td><td>Arg</td><td>Thr</td><td>Arg</td><td>80 Tyr</td>

<td>Tyr</td><td>Glu</td><td>Gin</td><td>val</td><td>85 How much</td><td>Gin</td><td>ala</td><td>Underworld</td><td>Leu</td><td>90 Gin</td><td>Cheese</td><td>How much</td><td>Gly</td><td>val</td><td>95 Pro</td><td>Leu</td>

<td>Glu</td><td>Arg</td><td>Leu</td><td>100 Arg</td><td>phe</td><td>How much</td><td>Arg</td><td>Gly</td><td>105 Thr</td><td>Glu</td><td>phe</td><td>Gin</td><td>Leu</td><td>110 Cheese</td><td>Ly3</td><td>Glu</td>

<td>Tyr</td><td>Thr</td><td>115 Leu</td><td>Asp</td><td>val</td><td>Tyr</td><td>Arg</td><td>120 Leu</td><td>Cheese</td><td>Cheese</td><td>val</td><td>val</td><td>125 Thr</td><td>Gin</td><td>His</td><td>Asp</td>

<td>ala</td><td>130 lys</td><td>lys</td><td>ala</td><td>Gly</td><td>ala</td><td>135 Glu</td><td>val</td><td>val</td><td>lys</td><td>Gin</td><td>140 val</td><td>Glu</td><td>His</td><td>Pro</td><td>Leu</td>

<td>145 Leu</td><td>Cheese</td><td>Gly</td><td>Leu</td><td>Leu</td><td>150 Tyr</td><td>pro</td><td>Gly</td><td>Leu</td><td>Gin</td><td>155 ala</td><td>Leu</td><td>Asp</td><td>Glu</td><td>Glu</td><td>160 Tyr</td>

<td>Leu</td><td>lys</td><td>val</td><td>Asp</td><td>165 ala</td><td>Gin</td><td>phe</td><td>Gly</td><td>Gly</td><td>170 val</td><td>Asp</td><td>Gin</td><td>Arg</td><td>lys</td><td>175 how much</td><td>phe</td>

<td>Thr</td><td>phe</td><td>ala</td><td>180 Glu</td><td>lys</td><td>Tyr</td><td>Leu</td><td>Pro</td><td>185 ala</td><td>Leu</td><td>Gly</td><td>Tyr</td><td>ala</td><td>190 lys</td><td>Arg</td><td>How much</td>

<td>His</td><td>Leu</td><td>195 Underworld</td><td>own</td><td>Pro</td><td>Underworld</td><td>val</td><td>200 Pro</td><td>Gly</td><td>Leu</td><td>Thr</td><td>Gly</td><td>205 ala</td><td>lys</td><td>Underworld</td><td>Cheese</td>

<td>Cheese</td><td>210 Cheese</td><td>Glu</td><td>Glu</td><td>Glu</td><td>Cheese</td><td>215 lys</td><td>How much</td><td>Asp</td><td>Leu</td><td>Leu</td><td>220 Asp</td><td>cheese</td><td>Pro</td><td>ala</td><td>Asp</td>

<td>22S val</td><td>lys</td><td>lys</td><td>lys</td><td>Leu</td><td>230 lys</td><td>lys</td><td>ala</td><td>phe</td><td>cys</td><td>235 Glu</td><td>Pro</td><td>Gly</td><td>own</td><td>val</td><td>240 Glu</td>

<td>own</td><td>own</td><td>Gly</td><td>val</td><td>245 Leu</td><td>Cheese</td><td>phe</td><td>val</td><td>Arg</td><td>250 His</td><td>val</td><td>Leu</td><td>phe</td><td>Pro</td><td>255 Leu</td><td>Lye</td>

<td>Cheese</td><td>Glu</td><td>phe</td><td>260 val</td><td>val</td><td>Leu</td><td>Arg</td><td>Asp</td><td>265 Glu</td><td>lys</td><td>phe</td><td>Gly</td><td>Gly</td><td>270 own</td><td>lys</td><td>Thr</td>

<td>Tyr</td><td>Thr</td><td>275 Asp</td><td>phe</td><td>Glu</td><td>Thr</td><td>Leu</td><td>280 Glu</td><td>lys</td><td>Asp</td><td>phe</td><td>ala</td><td>285 Glu</td><td>Glu</td><td>Leu</td><td>val</td>

<td>His</td><td>290 Pro</td><td>Gly</td><td>Asp</td><td>Leu</td><td>lys</td><td>295 ala</td><td>Cheese</td><td>val</td><td>Glu</td><td>lys</td><td>300 ala</td><td>Leu</td><td>own</td><td>lys</td><td>Leu</td>

<td>305 Leu</td><td>His</td><td>Pro</td><td>How much</td><td>Arg</td><td>310 Glu</td><td>lys</td><td>phe</td><td>own</td><td>Cheese</td><td>315 Pro</td><td>Glu</td><td>Underworld</td><td>lys</td><td>lys</td><td>320 Leu</td>

<td>Cheese</td><td>own</td><td>Asp</td><td>ala</td><td>325 Tyr</td><td>Pro</td><td>Asp</td><td>ala</td><td>Cheese</td><td>330 lys</td><td>Gin</td><td>lys</td><td>Cheese</td><td>val</td><td>335 Pro</td><td>lys</td>

<td>Gly</td><td>Cheese</td><td>Thr</td><td>340 lys</td><td>own</td><td>Cheese</td><td>Gly</td><td>Thr</td><td>345 Glu</td><td>Glu</td><td>How much</td><td>Asp</td><td>Pro</td><td>350 Cheese</td><td>Leu</td><td>Leu</td>

<td>Asp</td><td>Leu</td><td>355 Arg</td><td>val</td><td>Gly</td><td>lys</td><td>How much</td><td>360 Leu</td><td>Cheese</td><td>VAL</td><td>Cheese</td><td>Gin</td><td>365 His</td><td>Pro</td><td>Asp</td><td>ala</td>

<td>Asp</td><td>370 Cheese</td><td>Leu</td><td>Tyr</td><td>val</td><td>Glu</td><td>375 Cheese</td><td>val</td><td>Asp</td><td>val</td><td>Gly</td><td>380 Glu</td><td>ala</td><td>own</td><td>Pro</td><td>Arg</td>

<td>33S Cys</td><td>val</td><td>val</td><td>Cheese</td><td>Gly</td><td>390 Leu</td><td>val</td><td>Gin</td><td>Tyr</td><td>val</td><td>395 Pro</td><td>Cheese</td><td>Asp</td><td>Gin</td><td>Leu</td><td>400 Leu</td>

<td>Gly</td><td>Arg</td><td>Cheese</td><td>val</td><td>405 val</td><td>Leu</td><td>Leu</td><td>Cys</td><td>own</td><td>410 Leu</td><td>lys</td><td>Pro</td><td>Gin</td><td>lys</td><td>415 Underworld</td><td>Arg</td>

<td>Gly</td><td>How much</td><td>Glu</td><td>420 Cheese</td><td>Gin</td><td>Gly</td><td>Underworld</td><td>Leu</td><td>425 Leu</td><td>CYS</td><td>ala</td><td>Cheese</td><td>Thr</td><td>430 Glu</td><td>Gly</td><td>Glu</td>

<td>Gin</td><td>lys</td><td>435 Gin</td><td>val</td><td>Glu</td><td>Pro</td><td>Leu</td><td>440 Asp</td><td>Pro</td><td>Pro</td><td>cheese</td><td>Gly</td><td>445 Cheese</td><td>ala</td><td>pro</td><td>Gly</td>

<td>Glu</td><td>450 Arg</td><td>How much</td><td>Tyr</td><td>How much</td><td>Glu</td><td>455 Gly</td><td>Tyr</td><td>Glu</td><td>own</td><td>Gly</td><td>460 Glu</td><td>Pro</td><td>Glu</td><td>Gly</td><td>Glu</td>

<td>465 Leu</td><td>lys</td><td>Pro</td><td>lys</td><td>lys</td><td>4 70 Bald</td><td>val</td><td>phe</td><td>Glu</td><td>lys</td><td>475 Leu</td><td>Gin</td><td>val</td><td>Asp</td><td>phe</td><td>480 Arg</td>

<td>How much</td><td>Cheese</td><td>Asp</td><td>Asp</td><td>485 Leu</td><td>Cys</td><td>ala</td><td>Gin</td><td>Trp</td><td>490 lys</td><td>Gly</td><td>lys</td><td>own</td><td>phe</td><td>495 Leu</td><td>Thr</td>

<td>lys</td><td>Leu</td><td>Gly</td><td>500 cheese</td><td>val</td><td>Thr</td><td>Cys</td><td>lys</td><td>50S Thr</td><td>Leu</td><td>Arg</td><td>Gly</td><td>Gly</td><td>510 Cheese</td><td>how much</td><td>Gly</td>

515 520 525 &lt;210&gt; 55 &lt;211&gt; 402 &lt;212&gt; PRT &lt;213&gt; Helicobacter pylori &lt;400&gt; 55

-124-

<td>Underworld 1</td><td>Glu</td><td>Gin</td><td>lys</td><td>How much 5</td><td>ala</td><td>How much</td><td>ala</td><td>Leu</td><td>lys 10</td><td>Glu</td><td>How much</td><td>ala</td><td>Arg</td><td>Gly 15</td><td>Thr</td>

<td>own</td><td>Glu</td><td>How much</td><td>How much twenty</td><td>Gly</td><td>Leu</td><td>Glu</td><td>Tyr</td><td>How much 25</td><td>Glu</td><td>lys</td><td>Leu</td><td>val</td><td>Arg thirty</td><td>lys</td><td>Tyr</td>

<td>Tyr</td><td>Glu</td><td>Thr 35</td><td>own</td><td>Glu</td><td>Arg</td><td>phe</td><td>How much 40</td><td>val</td><td>lys</td><td>ala</td><td>Gly</td><td>phe 45</td><td>Asp</td><td>Pro</td><td>Thr</td>

<td>ala</td><td>Pro 50</td><td>Asp</td><td>Leu</td><td>His</td><td>Leu</td><td>Gly 55</td><td>His</td><td>Thr</td><td>val</td><td>Leu</td><td>how much 60</td><td>Gin</td><td>lys</td><td>Leu</td><td>ala</td>

<td>Leu 65</td><td>Leu</td><td>Gin</td><td>Gin</td><td>Tyr</td><td>Gly 70</td><td>ala</td><td>Arg</td><td>val</td><td>lys</td><td>phe 75</td><td>Leu</td><td>How much</td><td>Gly</td><td>ASp</td><td>phe BECAUSE</td>

<td>Thr</td><td>ala</td><td>Underworld</td><td>How much</td><td>Gly 85</td><td>Asp</td><td>Pro</td><td>Thr</td><td>Gly</td><td>lys 90</td><td>own</td><td>Glu</td><td>Thr</td><td>Arg</td><td>lys 95</td><td>Pro</td>

<td>Leu</td><td>own</td><td>Arg</td><td>Glu 100</td><td>Gin</td><td>val</td><td>Leu</td><td>Glu</td><td>own 105</td><td>ala</td><td>lys</td><td>Thr</td><td>Tyr</td><td>Glu 110</td><td>Glu</td><td>Gin</td>

<td>How much</td><td>Tyr</td><td>lys 115</td><td>How much</td><td>Leu</td><td>Asp</td><td>Glu</td><td>lys 120</td><td>His</td><td>Thr</td><td>Glu</td><td>val</td><td>Cys 12 5</td><td>phe</td><td>own</td><td>Cheese</td>

<td>Thr</td><td>Trp 130</td><td>Leu</td><td>Asp</td><td>ala</td><td>Leu</td><td>Gly 135</td><td>ala</td><td>lys</td><td>Gly</td><td>Underworld</td><td>How much 140</td><td>Glu</td><td>Leu</td><td>Cys</td><td>ala</td>

<td>lys 14S</td><td>phe</td><td>Cheese</td><td>val</td><td>ala</td><td>Arg 150</td><td>mec</td><td>Leu</td><td>Glu</td><td>Arg</td><td>Asp 155</td><td>Asp</td><td>phe</td><td>Thr</td><td>lys</td><td>Arg 160</td>

<td>Tyr</td><td>lys</td><td>Glu</td><td>own</td><td>Arg 165</td><td>Pro</td><td>How much</td><td>Cheese</td><td>How much</td><td>val 170</td><td>Glu</td><td>phe</td><td>Leu</td><td>Tyr</td><td>Pro 175</td><td>Leu</td>

<td>Leu</td><td>Gin</td><td>Gly</td><td>Tyr 180</td><td>Asp</td><td>Cheese</td><td>val</td><td>ala</td><td>Underworld 185</td><td>Asp</td><td>ala</td><td>Asp</td><td>How much</td><td>Siu 190</td><td>Leu</td><td>Gly</td>

<td>Gly</td><td>own</td><td>Asp 19S</td><td>Gin</td><td>lys</td><td>phe</td><td>own</td><td>Leu 200</td><td>Leu</td><td>val</td><td>Gly</td><td>Arg</td><td>phe 205</td><td>Leu</td><td>Gin</td><td>Arg</td>

<td>ala</td><td>Tyr 210</td><td>Gly</td><td>Leu</td><td>own</td><td>lys</td><td>Glu 21S</td><td>Gin</td><td>Cheese</td><td>val</td><td>How much</td><td>Thr 220</td><td>Underworld</td><td>Pro</td><td>Leu</td><td>Leu</td>

<td>Glu 22S</td><td>Gly</td><td>Leu</td><td>Asp</td><td>Gly</td><td>val 230</td><td>Gin</td><td>lys</td><td>Underworld</td><td>Cheese</td><td>lys 235</td><td>Cheese</td><td>Leu</td><td>Gly</td><td>own</td><td>Tyr 240</td>

<td>val</td><td>Gly</td><td>How much</td><td>Thr</td><td>Glu 245</td><td>Glu</td><td>Pro</td><td>own</td><td>ala</td><td>Underworld 250</td><td>phe</td><td>Gly</td><td>lys</td><td>How much</td><td>Underworld 255</td><td>Cheese</td>

<td>val</td><td>Cheese</td><td>Asp</td><td>Asp 260</td><td>Leu</td><td>Underworld</td><td>Trp</td><td>Arg</td><td>Tyr 265</td><td>Tyr</td><td>Thr</td><td>Leu</td><td>Leu</td><td>Cheese 270</td><td>Thr</td><td>lys</td>

<td>Thr</td><td>Leu</td><td>Glu 275</td><td>Glu</td><td>How much</td><td>Glu</td><td>Asp</td><td>Leu 280</td><td>lys</td><td>His</td><td>Gly</td><td>How much</td><td>Leu 285</td><td>ASN</td><td>Gin</td><td>Thr</td>

<td>Leu</td><td>His 290</td><td>Pro</td><td>lys</td><td>ala</td><td>val</td><td>lys 295</td><td>Glu</td><td>Asp</td><td>Leu</td><td>ala</td><td>Cheese 300</td><td>Glu</td><td>How much</td><td>val</td><td>ala</td>

<td>Arg 305</td><td>Tyr</td><td>Tyr</td><td>Asp</td><td>own</td><td>Asp 310</td><td>Gin</td><td>ala</td><td>How much</td><td>lys</td><td>ala 315</td><td>lys</td><td>Glu</td><td>Gin</td><td>phe</td><td>Cheese 320</td>

<td>lys</td><td>val</td><td>phe</td><td>Cheese</td><td>ala 325</td><td>own</td><td>Leu</td><td>Leu</td><td>Pro</td><td>Glu 330</td><td>How much</td><td>Leu</td><td>Cheese</td><td>Glu</td><td>Cheese 335</td><td>Asp</td>

<td>phe</td><td>Asp</td><td>Glu</td><td>Gly 340</td><td>val</td><td>Gly</td><td>How much</td><td>Leu</td><td>Asp 345</td><td>val</td><td>Leu</td><td>lys</td><td>Gin</td><td>How much 350</td><td>Gly</td><td>phe</td>

<td>Cys</td><td>Pro</td><td>Cheese 355</td><td>Thr</td><td>Cheese</td><td>Gin</td><td>ala</td><td>Arg 360</td><td>Arg</td><td>Asp</td><td>How much</td><td>Gin</td><td>Gly 365</td><td>Gly</td><td>Gly</td><td>val</td>

<td>lys</td><td>how much 370</td><td>aan</td><td>Gin</td><td>Glu</td><td>val</td><td>How much 375</td><td>lys</td><td>own</td><td>Glu</td><td>Cheese</td><td>Tyr 380</td><td>Arg</td><td>phe</td><td>val</td><td>lys</td>

<td>Gly 385 How much</td><td>own own</td><td>Tyr</td><td>val</td><td>How much</td><td>Gin 390</td><td>Leu</td><td>Gly</td><td>lys</td><td>lys</td><td>Arg 395</td><td>phe</td><td>Underworld</td><td>lys</td><td>Leu</td><td>own 400</td>

&lt;210&gt; 56 &lt;211&gt; 447 &lt;212&gt; PRT &lt;213&gt; Caenorhabditis elegans &lt;400&gt; 56

<td colspan="5"></td><td colspan="11"> -125-</td>

<td>Underworld</td><td>phe</td><td>own</td><td>cheese</td><td>lys</td><td>Arg</td><td>How much</td><td>Gly</td><td>own</td><td>val</td><td>ala</td><td>Leu</td><td>lys</td><td>Thr</td><td>val</td><td>Arg</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>ala</td><td>Pro</td><td>Arg</td><td>Glu</td><td>Cheese</td><td>Cheese</td><td>phe</td><td>val</td><td>Asp</td><td>Tyr</td><td>How much</td><td>Thr</td><td>Asp</td><td>Leu</td><td>own</td><td>ala</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Arg</td><td>lys</td><td>Gin</td><td>Leu</td><td>Gin</td><td>His</td><td>Cheese</td><td>Tyr</td><td>Pro</td><td>Thr</td><td>Asp</td><td>Leu</td><td>Leu</td><td>Cheese</td><td>lys</td><td>Cys</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Cheese</td><td>Glu</td><td>Asp</td><td>Leu</td><td>Arg</td><td>Gin</td><td>Leu</td><td>Pro</td><td>Pro</td><td>Tyr</td><td>val</td><td>Tyr</td><td>ala</td><td>Gly</td><td>phe</td><td>Asp</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Pro</td><td>Thr</td><td>ala</td><td>Glu</td><td>Cheese</td><td>Leu</td><td>His</td><td>How much</td><td>Gly</td><td>own</td><td>Leu</td><td>Leu</td><td>How much</td><td>Leu</td><td>val</td><td>own</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Leu</td><td>How much</td><td>Arg</td><td>ala</td><td>Gin</td><td>Gin</td><td>phe</td><td>Gly</td><td>How much</td><td>Arg</td><td>Pro</td><td>How much</td><td>ala</td><td>Leu</td><td>How much</td><td>Gly</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Glu</td><td>phe</td><td>Thr</td><td>ala</td><td>Cheese</td><td>How much</td><td>Gly</td><td>Asp</td><td>Pro</td><td>Cheese</td><td>Gly</td><td>lys</td><td>lys</td><td>Cheese</td><td>ol</td><td>Arg</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Gly</td><td>Leu</td><td>Leu</td><td>ala</td><td>Gly</td><td>Asp</td><td>val</td><td>How much</td><td>Underworld</td><td>His</td><td>own</td><td>Cheese</td><td>Arg</td><td>lys</td><td>val</td><td>Thr</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>aap</td><td>Gin</td><td>How much</td><td>Cys</td><td>lys</td><td>How much</td><td>phe</td><td>Glu</td><td>own</td><td>ala</td><td>Pro</td><td>Gly</td><td>Cheese</td><td>Cheese</td><td>Glu</td><td>lys</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Pro</td><td>How much</td><td>How much</td><td>val</td><td>own</td><td>own</td><td>own</td><td>Asp</td><td>Trp</td><td>Leu</td><td>Gly</td><td>lys</td><td>How much</td><td>Cheese</td><td>Leu</td><td>Arg</td>

<td> 14 5</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>aap</td><td>phe</td><td>Leu</td><td>Arg</td><td>Glu</td><td>Cys</td><td>lys</td><td>own</td><td>Underworld</td><td>Gin</td><td>val</td><td>Gly</td><td>lys</td><td>Underworld</td><td>Leu</td><td>Arg</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 17 5</td><td></td>

<td>Underworld</td><td>Aen</td><td>Thr</td><td>How much</td><td>Lya</td><td>own</td><td>Arg</td><td>Leu</td><td>Glu</td><td>val</td><td>Gly</td><td>Leu</td><td>Cheese</td><td>Tyr</td><td>Thr</td><td>Glu</td>

<td></td><td></td><td></td><td>ISO</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>phe</td><td>Cheese</td><td>Tyr</td><td>Gin</td><td>Thr</td><td>Underworld</td><td>Gin</td><td>ala</td><td>phe</td><td>Asp</td><td>Trp</td><td>Tyr</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>Glu</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>lys</td><td>Tyr</td><td>Gly</td><td>Cys</td><td>Arg</td><td>phe</td><td>Gin</td><td>Leu</td><td>Gly</td><td>Gly</td><td>Tyr</td><td>Asp</td><td>Gin</td><td>Leu</td><td>Gly</td><td>His</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Leu</td><td>Asp</td><td>phe</td><td>Gly</td><td>ala</td><td>His</td><td>Tyr</td><td>How much</td><td>lys</td><td>lys</td><td>Underworld</td><td>Underworld</td><td>own</td><td>Gin</td><td>ala</td><td>phe</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>ala</td><td>ala</td><td>Gly</td><td>val</td><td>Cys</td><td>phe</td><td>Pro</td><td>How much</td><td>Leu</td><td>Thr</td><td>Asp</td><td>Cheese</td><td>Thr</td><td>Gly</td><td>ala</td><td>lys</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

<td>Leu</td><td>Gly</td><td>lys</td><td>Cheese</td><td>Glu</td><td>Gly</td><td>Gly</td><td>Gly</td><td>ala</td><td>Leu</td><td>Trp</td><td>Leu</td><td>Asp</td><td>ala</td><td>Thr</td><td>lys</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>Thr</td><td>Cheese</td><td>Pro</td><td>phe</td><td>His</td><td>phe</td><td>Tyr</td><td>Gin</td><td>phe</td><td>phe</td><td>ala</td><td>Gin</td><td>Leu</td><td>His</td><td>Asp</td><td>Asp</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>LyS</td><td>ala</td><td>Glu</td><td>Glu</td><td>Leu</td><td>Leu</td><td>Leu</td><td>Leu</td><td>phe</td><td>Cheese</td><td>Leu</td><td>Gin</td><td>Asp</td><td>How much</td><td>Glu</td><td>His</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>How much</td><td>Arg</td><td>Asp</td><td>val</td><td>Leu</td><td>lys</td><td>own</td><td>His</td><td>Arg</td><td>cheese</td><td>own</td><td>Leu</td><td>Gly</td><td>Gin</td><td>Trp</td><td>How much</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td>31S</td><td></td><td></td><td></td><td></td><td> 320</td>

<td>ala</td><td>Gin</td><td>Arg</td><td>Glu</td><td>Leu</td><td>ala</td><td>ala</td><td>Glu</td><td>How much</td><td>Thr</td><td>Arg</td><td>How much</td><td>val</td><td>His</td><td>Gly</td><td>lys</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td>

<td>Glu</td><td>Gly</td><td>Leu</td><td>Glu</td><td>val</td><td>ala</td><td>Underworld</td><td>Arg</td><td>Cys</td><td>Thr</td><td>lys</td><td>ala</td><td>Underworld</td><td>phe</td><td>Gly</td><td>ala</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td>

<td>lys</td><td>lys</td><td>ala</td><td>aap</td><td>Leu</td><td>set</td><td>Gly</td><td>Leu</td><td>Cheese</td><td>Arg</td><td>Cheese</td><td>Glu</td><td>val</td><td>Leu</td><td>Gin</td><td>Leu</td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td>

<td>phe</td><td>Arg</td><td>Thr</td><td>Thr</td><td>How much</td><td>Asp</td><td>Leu</td><td>lys</td><td>lys</td><td>Glu</td><td>own</td><td>val</td><td>ala</td><td>Thr</td><td>Underworld</td><td>Gly</td>

<td></td><td> 370</td><td></td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td>

<td>Gin</td><td>Leu</td><td>ala</td><td>Asp</td><td>ala</td><td>Cheese</td><td>Arg</td><td>ltl *</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>lys</td><td>Gly</td><td>His</td><td>Leu</td><td>Leu</td>

<td> 385</td><td></td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td>

<td>Underworld</td><td>Gin</td><td>Gin</td><td>Gly</td><td>ala</td><td>phe</td><td>Cheese</td><td>val</td><td>own</td><td>Gly</td><td>Glu</td><td>lys</td><td>lys</td><td>Arg</td><td>Cheese</td><td>Pro</td>

<td></td><td></td><td></td><td></td><td> 405</td><td></td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td>

<td>Cheese</td><td>Glu</td><td>Cheese</td><td>How much</td><td>ala</td><td>Asp</td><td>val</td><td>phe</td><td>Leu</td><td>Glu</td><td>own</td><td>ala</td><td>Cheese</td><td>Asp</td><td>Leu</td><td>Thr</td>

<td></td><td></td><td></td><td> 420</td><td></td><td></td><td></td><td></td><td> 425</td><td></td><td></td><td></td><td></td><td> 430</td><td></td><td></td>

<td>Leu</td><td>val</td><td>Cys</td><td>Trp</td><td>Gly</td><td>lys</td><td>Arg</td><td>Gly</td><td>Tyr</td><td>Gin</td><td>Leu</td><td>val</td><td>Arg</td><td>Trp</td><td>val</td><td></td>

435 440 445 &lt;210&gt; 57 &lt;211&gt; 412 &lt;212&gt; PRT &lt;213&gt; Pseudomonas aeruginosa &lt;400&gt; 57

-126-

<td>mec 1</td><td colspan="2">Val cheese</td><td colspan="4">Pro Thr His Gin S</td><td colspan="3">Gin Asp Leu 10</td><td colspan="2">how much Ala</td><td colspan="2">Leu Leu</td><td>Glu 15</td><td>Glu</td>

<td>Arg</td><td>Gly</td><td>phe</td><td>val</td><td>His</td><td>Gin</td><td>Cy3</td><td>Thr</td><td>Asp</td><td>Arg</td><td>Asp</td><td>Gly</td><td>Leu</td><td>ala</td><td>ala</td><td>His</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Leu</td><td>ala</td><td>ala</td><td>Gly</td><td>Pro</td><td>ala</td><td>Thr</td><td>ala</td><td>Tyr</td><td>Leu</td><td>Gly</td><td>phe</td><td>Asp</td><td>ala</td><td>Thr</td><td>ala</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Asp</td><td>Cheese</td><td>Leu</td><td>His</td><td>val</td><td>Gly</td><td>His</td><td>Leu</td><td>Gin</td><td>Gly</td><td>Leu</td><td>Underworld</td><td>Leu</td><td>Underworld</td><td>Arg</td><td>Trp</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Leu</td><td>Gin</td><td>lys</td><td>ala</td><td>Gly</td><td>His</td><td>Arg</td><td>Pro</td><td>Leu</td><td>Leu</td><td>Leu</td><td>How much</td><td>Gly</td><td>Gly</td><td>ala</td><td>Thr</td>

<td> 55</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Thr</td><td>Arg</td><td>How much</td><td>Gly</td><td>Asp</td><td>Pro</td><td>Cheese</td><td>phe</td><td>Arg</td><td>Asp</td><td>cheese</td><td>Cheese</td><td>Arg</td><td>Pro</td><td>how much</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Thr</td><td>Glu</td><td>ala</td><td>Gin</td><td>How much</td><td>Gin</td><td>ala</td><td>own</td><td>How much</td><td>Asp</td><td>Gly</td><td>How much</td><td>ala</td><td>Arg</td><td>val</td><td>phe</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Cheese</td><td>Arg</td><td>Tyr</td><td>val</td><td>Glu</td><td>Leu</td><td>His</td><td>Asp</td><td>Asp</td><td>Cheese</td><td>Leu</td><td>val</td><td>own</td><td>own</td><td>ala</td><td>Glu</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Trp</td><td>Leu</td><td>Asp</td><td>Gly</td><td>vai</td><td>Gly</td><td>Tyr</td><td>Leu</td><td>Glu</td><td>phe</td><td>Leu</td><td>Asp</td><td>Arg</td><td>val</td><td>Gly</td><td>Arg</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>His</td><td>phe</td><td>Cheese</td><td>How much</td><td>own</td><td>Arg</td><td>Leu</td><td>Leu</td><td>Thr</td><td>phe</td><td>Asp</td><td>ala</td><td>How much</td><td>Arg</td><td>Gin</td><td>Arg</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Leu</td><td>Asp</td><td>Arg</td><td>Glu</td><td>His</td><td>Cheese</td><td>Leu</td><td>cheese</td><td>phe</td><td>Leu</td><td>Glu</td><td>phe</td><td>Gly</td><td>Tyr</td><td>Thr</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Leu</td><td>Gin</td><td>ala</td><td>Tyr</td><td>Asp</td><td>phe</td><td>val</td><td>Glu</td><td>Leu</td><td>Cheese</td><td>Arg</td><td>Arg</td><td>Arg</td><td>Gly</td><td>Cys</td><td>Thr</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Leu</td><td>Gin</td><td>Leu</td><td>Gly</td><td>Gly</td><td>ala</td><td>Asp</td><td>Gin</td><td>Trp</td><td>ala</td><td>own</td><td>How much</td><td>How much</td><td>own</td><td>Gly</td><td>val</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td>20S</td><td></td><td></td><td></td>

<td>Glu</td><td>Leu</td><td>Cheese</td><td>Arg</td><td>Arg</td><td>Gin</td><td>Gly</td><td>Gly</td><td>ala</td><td>Gin</td><td>Leu</td><td>phe</td><td>Gly</td><td>Leu</td><td>Thr</td><td>Underworld</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Pro</td><td>Leu</td><td>Leu</td><td>ala</td><td>Thr</td><td>Cheese</td><td>Asp</td><td>Gly</td><td>Arg</td><td>lys</td><td>Underworld</td><td>Gly</td><td>lys</td><td>9ER</td><td>ala</td><td>Gin</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>Gly</td><td>ala</td><td>val</td><td>Trp</td><td>Leu</td><td>own</td><td>ala</td><td>Glu</td><td>Arg</td><td>Leu</td><td>ala</td><td>Pro</td><td>phe</td><td>Asp</td><td>phe</td><td>Trp</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

<td>Gin</td><td>phe</td><td>Trp</td><td>Arg</td><td>own</td><td>Cys</td><td>Asp</td><td>Asp</td><td>Arg</td><td>Asp</td><td>val</td><td>Gly</td><td>Arg</td><td>phe</td><td>Leu</td><td>ala</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td>26S</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>Leu</td><td>phe</td><td>Cheese</td><td>Glu</td><td>Leu</td><td>Pro</td><td>Underworld</td><td>Asp</td><td>Glu</td><td>val</td><td>Arg</td><td>Arg</td><td>Leu</td><td>Gly</td><td>ala</td><td>Leu</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td rowspan="2">val</td><td></td><td> 285</td><td></td><td></td><td></td>

<td>Gin</td><td>Gly</td><td>ala</td><td>Glu</td><td>Leu</td><td>own</td><td>Glo</td><td>ala</td><td>lys</td><td>val</td><td>Leu</td><td>ala</td><td>own</td><td>ala</td><td>ala</td>

<td></td><td> 2 90</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>Thr</td><td>ala</td><td>Leu</td><td>ala</td><td>His</td><td>Gly</td><td>Glu</td><td>His</td><td>ala</td><td>ala</td><td>Arg</td><td>cheese</td><td>ala</td><td>ala</td><td>Asp</td><td>ala</td>

<td>30S</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

<td>ala</td><td>Arg</td><td>Gly</td><td>val</td><td>phe</td><td>ala</td><td>Asp</td><td>Gly</td><td>Thr</td><td>Arg</td><td>ASp</td><td>Cheese</td><td>Gly</td><td>Leu</td><td>Pro</td><td>val</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td>

<td>Underworld</td><td>lys</td><td>Leu</td><td>Cheese</td><td>Arg</td><td>ala</td><td>Arg</td><td>Leu</td><td>ala</td><td>Gin</td><td>Gly</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Thr</td><td>Asp</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td>

<td>Leu</td><td>Leu</td><td>Leu</td><td>Glu</td><td>His</td><td>ala</td><td>How much</td><td>Gin</td><td>Pro</td><td>Cheese</td><td>Arg</td><td>Cheese</td><td>ala</td><td>val</td><td>Arg</td><td>Arg</td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td>

<td>Leu</td><td>ala</td><td>ala</td><td>Gly</td><td colspan="2">Gly gly</td><td>Leu</td><td>Arg</td><td>Leu</td><td>Asp</td><td>Gly</td><td>Thr</td><td>Pro</td><td>val</td><td>Cheese</td><td>Asp</td>

<td></td><td> 370</td><td></td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td>

<td>Pro</td><td>Asp</td><td>Thr</td><td>Pro</td><td>Leu</td><td>ala</td><td>Gly</td><td>Glu</td><td>val</td><td>Asp</td><td>Gly</td><td>Leu</td><td>Arg</td><td>Leu</td><td>Cheese</td><td>Leu</td>

<td> 385</td><td></td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td>

<td>Gly</td><td>lys</td><td>lys</td><td>Gin</td><td>His</td><td>Leu</td><td>His</td><td>Leu</td><td>Arg</td><td>Leu</td><td>Glu</td><td>Asp</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td> 405</td><td></td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 58 &lt;211&gt; 399 &lt;212&gt; PRT &lt;213&gt; Pseudomonas aeruginosa &lt;400&gt; 58

-127-

<td rowspan="2">Underworld 1</td><td rowspan="2">lys</td><td colspan="2" rowspan="2">Val cheese</td><td colspan="6">Glu Glu Gln Leu Ala Leu</td><td rowspan="2">How much</td><td rowspan="2">Gln</td><td rowspan="2">Arg</td><td rowspan="2">Gly</td><td colspan="2" rowspan="2">Ala Asp 15</td>

<td colspan="2"> 5</td><td colspan="4"> 10</td>

<td>Glu</td><td>How much</td><td>Leu</td><td>val</td><td>Glu</td><td>ala</td><td>Glu</td><td>Leu</td><td>val</td><td>ala</td><td>lys</td><td>Leu</td><td>lys</td><td>Arg</td><td>Gly</td><td>Gln</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Pro</td><td>Leu</td><td>Arg</td><td>How much</td><td>lys</td><td>ala</td><td>Gly</td><td>phe</td><td>Asp</td><td>Pro</td><td>Thr</td><td>ala</td><td>Pro</td><td>Asp</td><td>Leu</td><td>His</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Leu</td><td>Gly</td><td>His</td><td>Thr</td><td>val</td><td>Leu</td><td>How much</td><td>own</td><td>lys</td><td>Leu</td><td>Arg</td><td>Gln</td><td>phe</td><td>Gln</td><td>Asp</td><td>Leu</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>His</td><td>Gln</td><td>val</td><td>How much</td><td>phe</td><td>Leu</td><td>How much</td><td>Gly</td><td>Asp</td><td>phe</td><td>Thr</td><td>Gly</td><td>Underworld</td><td>How much</td><td>Gly</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Asp</td><td>Pro</td><td>Cheese</td><td>Gly</td><td>lys</td><td>Cheese</td><td>val</td><td>Thr</td><td>Arg</td><td>Pro</td><td>Pro</td><td>Leu</td><td>Thr</td><td>Arg</td><td>Glu</td><td>Gln</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>val</td><td>Leu</td><td>Glu</td><td>own</td><td>ala</td><td>Glu</td><td>Thr</td><td>Tyr</td><td>lys</td><td>Cheese</td><td>Gln</td><td>val</td><td>phe</td><td>lys</td><td>How much</td><td>Leu</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Asp</td><td>Pro</td><td>ala</td><td>lys</td><td>Thr</td><td>Glu</td><td>val</td><td>ala</td><td>phe</td><td>own</td><td>Cheese</td><td>Thr</td><td>Trp</td><td>Underworld</td><td>Asp</td><td>Gln</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Leu</td><td>Thr</td><td>Pro</td><td>ala</td><td>Asp</td><td>phe</td><td>How much</td><td>Arg</td><td>Leu</td><td>ala</td><td>Cheese</td><td>Gln</td><td>Tyr</td><td>Thr</td><td>val</td><td>ala</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 14 0</td><td></td><td></td><td></td><td></td>

<td>Arg</td><td>Underworld</td><td>Leu</td><td>□ lu</td><td>Arg</td><td>Asp</td><td>Asp</td><td>phe</td><td>Cheese</td><td>lys</td><td>Arg</td><td>Tyr</td><td>ala</td><td>Cheese</td><td>own</td><td>Gln</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Pro</td><td>How much</td><td>ala</td><td>How much</td><td>His</td><td>Glu</td><td>phe</td><td>Leu</td><td>Tyr</td><td>Pro</td><td>Leu</td><td>val</td><td>Gln</td><td>Gly</td><td>Tyr</td><td>aap</td>

<td></td><td></td><td></td><td></td><td>16S</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Cheese</td><td>val</td><td>ala</td><td>Leu</td><td>lys</td><td>ala</td><td>Asp</td><td>val</td><td>Glu</td><td>Leu</td><td>Gly</td><td>Gly</td><td>Thr</td><td>Asp</td><td>Gln</td><td>lys</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td>IBS</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>phe</td><td>own</td><td>Leu</td><td>Leu</td><td>Underworld</td><td>Gly</td><td>Arg</td><td>Glu</td><td>Leu</td><td>Gln</td><td>Arg</td><td>ala</td><td>Tyr</td><td>Gly</td><td>Gln</td><td>Glu</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>ala</td><td>Gln</td><td>val</td><td>How much</td><td>Leu</td><td>Thr</td><td>ket</td><td>Pro</td><td>Leu</td><td>Leu</td><td>Glu</td><td>Gly</td><td>Leu</td><td>Asp</td><td>Gly</td><td>val</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>lys</td><td>lys</td><td>Underworld</td><td>Cheese</td><td>lys</td><td>Cheese</td><td>Leu</td><td>Gly</td><td>own</td><td>Tyr</td><td>How much</td><td>Gly</td><td>How much</td><td>Gln</td><td>Glu</td><td>ala</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>Pro</td><td>Gly</td><td>val</td><td>Underworld</td><td>Tyr</td><td>Cheese</td><td>lys</td><td>Leu</td><td>val</td><td>Cheese</td><td>How much</td><td>Pro</td><td>Asp</td><td>Thr</td><td>Leu</td><td>Underworld</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td>2S5</td><td></td>

<td>Trp</td><td>Arg</td><td>Tyr</td><td>phe</td><td>Glu</td><td>Leu</td><td>Leu</td><td>Cheese</td><td>phe</td><td>Arg</td><td>Cheese</td><td>Leu</td><td>Asp</td><td>Glu</td><td>How much</td><td>Asp</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>Cheese</td><td>phe</td><td>Arg</td><td>lys</td><td>Asp</td><td>val</td><td>Glu</td><td>ala</td><td>Gly</td><td>ala</td><td>own</td><td>Pro</td><td>Arg</td><td>Asp</td><td>How much</td><td>lys</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>how much</td><td>lys</td><td>Leu</td><td>ala</td><td>Glu</td><td>Glu</td><td>How much</td><td>val</td><td>ala</td><td>Arg</td><td>phe</td><td>His</td><td>Gly</td><td>Glu</td><td>Glu</td><td>ala</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>ala</td><td>ala</td><td>Cheese</td><td>ala</td><td>His</td><td>lys</td><td>Cheese</td><td>ala</td><td>Gly</td><td>A9n</td><td>Arg</td><td>Leu</td><td>lys</td><td>Glu</td><td>Gly</td><td>Glu</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

<td>Leu</td><td>Pro</td><td>Glu</td><td>Asp</td><td>Leu</td><td>Pro</td><td>Glu</td><td>How much</td><td>Glu</td><td>Leu</td><td>Cheese</td><td>Cheese</td><td>Pro</td><td>GILJ</td><td>Asp</td><td>Underworld</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td>

<td>Pro</td><td>val</td><td>ala</td><td>Cheese</td><td>val</td><td>Leu</td><td>own</td><td>lys</td><td>ala</td><td>Gly</td><td>Leu</td><td>val</td><td>lys</td><td>own</td><td>ala</td><td>ala</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td>

<td>ala</td><td>ala</td><td>Arg</td><td>Asp</td><td>Leu</td><td>Leu</td><td>Gly</td><td>ala</td><td>Gly</td><td>Cheese</td><td>val</td><td>lys</td><td>val</td><td>Asp</td><td>Gly</td><td>Gln</td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td>

<td>val</td><td>val</td><td>Asp</td><td>Arg</td><td>Thr</td><td>phe</td><td>Underworld</td><td>Leu</td><td>ala</td><td>Leu</td><td>Gly</td><td>Glu</td><td>Thr</td><td>Arg</td><td>val</td><td>phe</td>

<td></td><td> 370</td><td></td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td>

<td>Gln</td><td>ala</td><td>Gly</td><td>lys</td><td>lys</td><td>ala</td><td>phe</td><td>ala</td><td>Arg</td><td>How much</td><td>Thr</td><td>Leu</td><td>lys</td><td>ala</td><td>Glu</td><td></td>

38S 390 395 &lt;210&gt; 59 &lt;211&gt; 334 &lt;212&gt; PRT &lt;213&gt; Escherichia coli &lt;400&gt; 59

-128-

<td rowspan="2">Underworld 1</td><td colspan="2" rowspan="2">Thr Lys</td><td colspan="2" rowspan="2">Pro Ile 5</td><td rowspan="2">val</td><td colspan="9">Phe Cheese Gly Ala Gin Pro Cheese Gly Glu</td><td rowspan="2">Leu</td>

<td colspan="8"> 10</td><td> 15</td>

<td>Thr</td><td>How much</td><td>Gly</td><td>own</td><td>Tyr</td><td>Underworld</td><td>Gly</td><td>ala</td><td>Leu</td><td>Arg</td><td>Gin</td><td>Trp</td><td>val</td><td>own</td><td>Underworld</td><td>Gin</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td>2S</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Asp</td><td>Asp</td><td>Tyr</td><td>His</td><td>Cys</td><td>How much</td><td>Tyr</td><td>Cys</td><td>How much</td><td>val</td><td>Asp</td><td>Gin</td><td>His</td><td>ala</td><td>How much</td><td>Thr</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>val</td><td>Arg</td><td>Gin</td><td>Asp</td><td>ala</td><td>Gin</td><td>lys</td><td>Leu</td><td>Arg</td><td>lys</td><td>ala</td><td>Thr</td><td>Leu</td><td>Asp</td><td>Thr</td><td>Leu</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>ala</td><td>Leu</td><td>Tyr</td><td>Leu</td><td>ala</td><td>Cys</td><td>Gly</td><td>How much</td><td>Asp</td><td>Pro</td><td>Glu</td><td>lys</td><td>Cheese</td><td>Thr</td><td>He</td><td>phe</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>val</td><td>Gin</td><td>Cheese</td><td>His</td><td>val</td><td>Pro</td><td>Glu</td><td>His</td><td>ala</td><td>Gin</td><td>Leu</td><td>Gly</td><td>Trp</td><td>ala</td><td>Leu</td><td>own</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Cys</td><td>Tyr</td><td>Thr</td><td>Tyr</td><td>phe</td><td>Gly</td><td>Glu</td><td>Leu</td><td>Cheese</td><td>Arg</td><td>Underworld</td><td>Thr</td><td>Gin</td><td>phe</td><td>lys</td><td>Asp</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>lys</td><td>Cheese</td><td>ala</td><td>Arg</td><td>Tyr</td><td>ala</td><td>Glu</td><td>own</td><td>He</td><td>own</td><td>ala</td><td>Gly</td><td>Leu</td><td>phe</td><td>Asp</td><td>Tyr</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Pro</td><td>val</td><td>Leu</td><td>Underworld</td><td>ala</td><td>ala</td><td>Asp</td><td>how much</td><td>Leu</td><td>Leu</td><td>Tyr</td><td>Gin</td><td>Thr</td><td>own</td><td>Leu</td><td>val</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Pro</td><td>val</td><td>Gly</td><td>Glu</td><td>Asp</td><td>Gin</td><td>lys</td><td>Gin</td><td>His</td><td>Leu</td><td>Glu</td><td>Leu</td><td>Cheese</td><td>Arg</td><td>Asp</td><td>How much</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td>1SS</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>ala</td><td>Gin</td><td>Arg</td><td>phe</td><td>own</td><td>ala</td><td>Leu</td><td>Tyr</td><td>Gly</td><td>Glu</td><td>how much</td><td>phe</td><td>lys</td><td>val</td><td>Pro</td><td>Glu</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 17 0</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Pro</td><td>phe</td><td>He</td><td>Pro</td><td>lys</td><td>Cheese</td><td>Gly</td><td>ala</td><td>Arg</td><td>val</td><td>Underworld</td><td>Cheese</td><td>Leu</td><td>Leu</td><td>Glu</td><td>Pro</td>

<td></td><td></td><td></td><td>ISO</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Thr</td><td>lys</td><td>lys</td><td>Underworld</td><td>Cheese</td><td>lys</td><td>Cheese</td><td>Asp</td><td>Asp</td><td>own</td><td>Arg</td><td>own</td><td>own</td><td>val</td><td>How much</td><td>Gly</td>

<td></td><td></td><td>19S</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Leu</td><td>Leu</td><td>Glu</td><td>ASP</td><td>Pro</td><td>lys</td><td>Cheese</td><td>val</td><td>val</td><td>lys</td><td>lys</td><td>How much</td><td>lys</td><td>Arg</td><td>ala</td><td>val</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Thr</td><td>Asp</td><td>Cheese</td><td>Asp</td><td>Glu</td><td>Pro</td><td>Pro</td><td>val</td><td>vai</td><td>Arg</td><td>Tyr</td><td>Asp</td><td>val</td><td>Gin</td><td>own</td><td>lys</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>ala</td><td>Gly</td><td>val</td><td>Cheese</td><td>own</td><td>Leu</td><td>Leu</td><td>Asp</td><td>How much</td><td>Leu</td><td>Cheese</td><td>ala</td><td>val</td><td>Thr</td><td>Gly</td><td>Gin</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

<td>Cheese</td><td>How much</td><td>Pro</td><td>Glu</td><td>Leu</td><td>Glu</td><td>lys</td><td>Gin</td><td>phe</td><td>Glu</td><td>Gly</td><td>lys</td><td>Underworld</td><td>Tyr</td><td>Gly</td><td>His</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>Leu</td><td>lys</td><td>Gly</td><td>Glu</td><td>val</td><td>ala</td><td>Asp</td><td>ala</td><td>val</td><td>Cheese</td><td>Gly</td><td>Underworld</td><td>Leu</td><td>Thr</td><td>Glu</td><td>Leu</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td>28S</td><td></td><td></td><td></td>

<td>Gin</td><td>Glu</td><td>Arg</td><td>Tyr</td><td>His</td><td>Arg</td><td>phe</td><td>Arg</td><td>own</td><td>Asp</td><td>Glu</td><td>ala</td><td>phe</td><td>Leu</td><td>Gin</td><td>Gin</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>val</td><td>Underworld</td><td>lys</td><td>Asp</td><td>Gly</td><td>ala</td><td>Glu</td><td>Lya</td><td>ala</td><td>Cheese</td><td>ala</td><td>His</td><td>ala</td><td>Cheese</td><td>Arg</td><td>Thr</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td>31S</td><td></td><td></td><td></td><td></td><td> 320</td>

<td>Leu</td><td>lys</td><td>ala</td><td>val</td><td>Tyr</td><td>Glu</td><td>ala</td><td>How much</td><td>Gly</td><td>phe</td><td>val</td><td>ala</td><td>lys</td><td>Pro</td><td></td><td></td>

325 330 &lt;210&gt; 60 &lt;211&gt; 326 &lt;212&gt; PRT &lt;213&gt; Helicobacter pylori &lt;400&gt; 60

-129-

<td>Underworld 1</td><td>His</td><td>lys</td><td>lys</td><td>Arg 5</td><td>val</td><td>phe</td><td>Cheese</td>

<td>His</td><td>Leu</td><td>Gly</td><td>own twenty</td><td>Tyr</td><td>Leu</td><td>Gly</td><td>ala</td>

<td>Asp</td><td>Glu</td><td>Tyr 35</td><td>Glu</td><td>own</td><td>Leu</td><td>phe</td><td>Cys 40</td>

<td>Leu</td><td>Pro 50</td><td>How much</td><td>Asp</td><td>Pro</td><td>ala</td><td>phe 55</td><td>Leu</td>

<td>lys 65</td><td>Leu</td><td>Leu</td><td>Leu</td><td>ala</td><td>Cya 70</td><td>Gly</td><td>How much</td>

<td>How much</td><td>Gin</td><td>Cheese</td><td>Glu</td><td>val 05</td><td>Asp</td><td>Glu</td><td>His</td>

<td>Cys</td><td>Gin</td><td>val</td><td>Cheese 100</td><td>Underworld</td><td>Gly</td><td>Glu</td><td>Underworld</td>

<td>Gly</td><td>How much 10</td><td>Gin</td><td>Pro</td><td>Thr</td><td>Gly</td><td>Gin 15</td><td>How much</td>

<td>How much 25</td><td>lys</td><td>His</td><td>Trp</td><td>val</td><td>Glu thirty</td><td>Underworld</td><td>Gin</td>

<td>val</td><td>val</td><td>own</td><td>Cheese</td><td>Kis 45</td><td>ala</td><td>How much</td><td>Thr</td>

<td>lys</td><td>Cheese</td><td>Gin</td><td>Cheese 60</td><td>Tyr</td><td>Glu</td><td>Leu</td><td>val</td>

<td>Asp</td><td>Pro</td><td>lys 75</td><td>Gin</td><td>Cheese</td><td>Gly</td><td>Leu</td><td>phe 80</td>

<td>Pro</td><td>ala 90</td><td>Leu</td><td>ala</td><td>Trp</td><td>Leu</td><td>Leu 95</td><td>own</td>

<td>Gin 105</td><td>Arg</td><td>Underworld</td><td>Thr</td><td>Gin</td><td>phe 110</td><td>lys</td><td>Asp</td>

<td>lys</td><td>Cheese</td><td>Leu</td><td>lys</td><td>own</td><td>Pro</td><td>lys</td><td>Cheese</td><td>val</td><td>own</td><td>val</td><td>Gly</td><td>Leu</td><td>phe</td><td>own</td><td>Tyr</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Pro</td><td>how much</td><td>Leu</td><td>Underworld</td><td>ala</td><td>Cheese</td><td>Asp</td><td>How much</td><td>Leu</td><td>Leu</td><td>Tyr</td><td>Gin</td><td>Cheese</td><td>Asp</td><td>Leu</td><td>vai</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Pro</td><td>val</td><td>Gly</td><td>Glu</td><td>Asp</td><td>Gin</td><td>lys</td><td>Gin</td><td>His</td><td>Leu</td><td>Glu</td><td>Leu</td><td>Thr</td><td>Arg</td><td>own</td><td>how much</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>ala</td><td>Glu</td><td>lys</td><td>phe</td><td>own</td><td>Arg</td><td>Asp</td><td>phe</td><td>Gly</td><td>own</td><td>Cys</td><td>phe</td><td>lys</td><td>val</td><td>Pro</td><td>Glu</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Pro</td><td>Leu</td><td>How much</td><td>ala</td><td>lys</td><td>val</td><td>Gly</td><td>ala</td><td>Arg</td><td>val</td><td>Underworld</td><td>Gly</td><td>Leu</td><td colspan="2">Asp Asp</td><td>Pro</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>lys</td><td>val</td><td>lys</td><td>Underworld</td><td>Cheese</td><td>lys</td><td>Cheese</td><td>His</td><td>Gin</td><td>Gly</td><td>ala</td><td>own</td><td>His</td><td>ala</td><td>How much</td><td>phe</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Leu</td><td>Leu</td><td>Asp</td><td>Glu</td><td>Pro</td><td>Asp</td><td>How much</td><td>how much</td><td>val</td><td>lys</td><td>lys</td><td>How much</td><td>lys</td><td>lys</td><td>ala</td><td>ala</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Thr</td><td>ASp</td><td>cheese</td><td>Underworld</td><td>Gly</td><td>val</td><td>How much</td><td>ala</td><td>phe</td><td>Asp</td><td>Glu</td><td>lys</td><td>Arg</td><td>Glu</td><td>Gly</td><td>How much</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>phe</td><td>own</td><td>Leu</td><td>Leu</td><td>own</td><td>How much</td><td>Tyr</td><td>Underworld</td><td>Leu</td><td>Leu</td><td>Cheese</td><td>own</td><td>Glu</td><td>der</td><td>Pro</td><td>Glu</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

<td>own</td><td>How much</td><td>Glu</td><td>Glu</td><td>Arg</td><td>phe</td><td>lys</td><td>own</td><td>lys</td><td>Gly</td><td>Tyr</td><td>Gly</td><td>Asp</td><td>phe</td><td>lys</td><td>lys</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>Glu</td><td>Leu</td><td>ala</td><td>Glu</td><td>val</td><td>Underworld</td><td>How much</td><td>Gin</td><td>ala</td><td>Leu</td><td>lys</td><td>Pro</td><td>How much</td><td>Gin</td><td>Glu</td><td>Arg</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>Tyr</td><td>lys</td><td>Glu</td><td>How much</td><td>Cheese</td><td>Asp</td><td>Asp</td><td>Glu</td><td>val</td><td>lys</td><td>ala</td><td>How much</td><td>Leu</td><td>own</td><td>Gly</td><td>Gly</td>

<td></td><td> 293</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>ala</td><td>Glu</td><td>lys</td><td>ala</td><td>Arg</td><td>Pro</td><td>Leu</td><td>ala</td><td>Arg</td><td>ala</td><td>Thr</td><td>Tyr</td><td>Gin</td><td>lys</td><td>ala</td><td>lys</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

<td>Glu</td><td>Leu</td><td>Underworld</td><td>Gly</td><td>Leu</td><td>How much</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

325 &lt;210&gt; 61 &lt;211&gt; 361 &lt;212&gt; PRT &lt;213&gt; Schizosaccharomyces pombe &lt;400&gt; 61

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<td colspan="4">Mec Ala Lys Leu 1</td><td>Pro 5</td><td>lys</td><td colspan="2">How much thr</td><td colspan="3">Leu Leu cheese 10</td><td>Pro</td><td>His</td><td>Cheese</td><td>Arg 15</td><td>val</td>

<td>val</td><td>Cheese</td><td>Gly</td><td>How much</td><td>Gin</td><td>Pro</td><td>Thr</td><td>Gly</td><td>How much</td><td>Pro</td><td>His</td><td>How much</td><td>Gly</td><td>own</td><td>Tyr</td><td>Leu</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Gly</td><td>Cheese</td><td>Leu</td><td>lys</td><td>Gin</td><td>Trp</td><td>val</td><td>Gin</td><td>Leu</td><td>Gin</td><td>Glu</td><td>Glu</td><td>ala</td><td>ala</td><td>Arg</td><td>Thr</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Pro</td><td>phe</td><td>Cheese</td><td>lys</td><td>Cys</td><td>phe</td><td>phe</td><td>phe</td><td>val</td><td>ala</td><td>Asp</td><td>Leu</td><td>His</td><td>ala</td><td>Leu</td><td>Thr</td>

<td></td><td>SO</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>val</td><td>Pro</td><td>Gin</td><td>Asp</td><td>Pro</td><td>Leu</td><td>lys</td><td>phe</td><td>Arg</td><td>Gin</td><td>ala</td><td>Arg</td><td>Leu</td><td>Asp</td><td>Underworld</td><td>Leu</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td>BECAUSE</td>

<td>.Ala</td><td>ala</td><td>Leu</td><td>Leu</td><td>ala</td><td>How much</td><td>Gly</td><td>How much</td><td>own</td><td>Pro</td><td>Gin</td><td>lys</td><td>Cheese</td><td>Thr</td><td>Leu</td><td>phe</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>phe</td><td>Gin</td><td>Cheese</td><td>Asp</td><td>val</td><td>ala</td><td>Gin</td><td>His</td><td>Cheese</td><td>Glu</td><td>Leu</td><td>ala</td><td>Trp</td><td>Leu</td><td>Leu</td><td>ala</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Cys</td><td>Cheese</td><td>Thr</td><td>Cheese</td><td>Underworld</td><td>Gly</td><td>Gin</td><td>Leu</td><td>own</td><td>Arg</td><td>Underworld</td><td>Thr</td><td>Gin</td><td>Trp</td><td>lys</td><td>Cheese</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Ly3</td><td>Leu</td><td>His</td><td>Leu</td><td>His</td><td>Asp</td><td>His</td><td>Asp</td><td>Asp</td><td>Leu</td><td>Cheese</td><td>phe</td><td>Leu</td><td>Asp</td><td>ala</td><td>Cheese</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>ala</td><td>Thr</td><td>Cheese</td><td>Cheese</td><td>Thr</td><td>Arg</td><td>phe</td><td>own</td><td>Leu</td><td>Gly</td><td>Leu</td><td>phe</td><td>Cheese</td><td>Tyr</td><td>Pro</td><td>val</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Leu</td><td>Gin</td><td>ala</td><td>ala</td><td>Asp</td><td>How much</td><td>Leu</td><td>Leu</td><td>Tyr</td><td>Gly</td><td>ala</td><td>Thr</td><td>His</td><td>How much</td><td>Pro</td><td>val</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Gly</td><td>lys</td><td>Asp</td><td>Gin</td><td>cheese</td><td>Gin</td><td>His</td><td>val</td><td>Glu</td><td>Leu</td><td>Thr</td><td>Arg</td><td>Cheese</td><td>How much</td><td>ala</td><td>Arg</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Cheese</td><td>phe</td><td>Aen</td><td>Cheese</td><td>Cheese</td><td>Tyr</td><td>lys</td><td>Glu</td><td>lys</td><td>How much</td><td>Leu</td><td>Thr</td><td>val</td><td>Pro</td><td>Asp</td><td>How much</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>How much</td><td>Leu</td><td>own</td><td>Cheese</td><td>Cheese</td><td>Cheese</td><td>Cheese</td><td>How much</td><td>Underworld</td><td>ala</td><td>Leu</td><td>Cys</td><td>Gin</td><td>Pro</td><td>Glu</td><td>lys</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>lys</td><td>Underworld</td><td>Cheese</td><td>lys</td><td>Cheese</td><td>Asp</td><td>How much</td><td>own</td><td>Cheese</td><td>lys</td><td>own</td><td>Tyr</td><td>How much</td><td>Leu</td><td>Leu</td><td>Cheese</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>Asp</td><td>Cheese</td><td>Thr</td><td>Gly</td><td>Glu</td><td>How much</td><td>Arg</td><td>lys</td><td>lys</td><td>How much</td><td>Cheese</td><td>Arg</td><td>ala</td><td>Gin</td><td>Thr</td><td>Asp</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td>2S0</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

<td>own</td><td>How much</td><td>lys</td><td>Gly</td><td>How much</td><td>Thr</td><td>Tyr</td><td>Gly</td><td>Asp</td><td>Cheese</td><td>own</td><td>Arg</td><td>Pro</td><td>Gly</td><td>How much</td><td>own</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>own</td><td>Leu</td><td>How much</td><td>own</td><td>How much</td><td>phe</td><td>ala</td><td>ala</td><td>How much</td><td>Cheese</td><td>Asp</td><td>Cheese</td><td>Thr</td><td>Pro</td><td>Cheese</td><td>Asp</td>

<td></td><td></td><td>27S</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>how much</td><td>ala</td><td>Gin</td><td>ala</td><td>own</td><td>ala</td><td>Cheese</td><td>Cys</td><td>Cheese</td><td>own</td><td>ala</td><td>Glu</td><td>phe</td><td>lys</td><td>Glu</td><td>lys</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td>29S</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>val</td><td>Cheese</td><td>Cheese</td><td>ala</td><td>How much</td><td>How much</td><td>Arg</td><td>Cys</td><td>Leu</td><td>Gin</td><td>Pro</td><td>How much</td><td>Cheese</td><td>Thr</td><td>Cheese</td><td>phe</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

<td>own</td><td>Glu</td><td>Trp</td><td>Arg</td><td>Gin</td><td>own</td><td>Arg</td><td>Glu</td><td>Leu</td><td>Leu</td><td>Arg</td><td>Asp</td><td>How much</td><td>ala</td><td>lys</td><td>lys</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td>

<td>Gly</td><td>ala</td><td>Glu</td><td>Glu</td><td>ala</td><td>val</td><td>ala</td><td>Glu</td><td>ala</td><td>Cheese</td><td>Cheese</td><td>Cys</td><td>Underworld</td><td>HI3</td><td>lys</td><td>Leu</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td>

<td>lys</td><td>Thr</td><td>Leu</td><td>Thr</td><td>Gly</td><td>Leu</td><td>Cheese</td><td>val</td><td>Tyr</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 62 &lt;211&gt; 665 &lt;212&gt; PRT &lt;213&gt; Drosophila melanogaster &lt;400&gt; 62

Mec Phe Arg Phe Gly Lys Leu His Arg Ser Leu Ser Lye Val Ala Lys

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<td>1 lys</td><td>Gly</td><td>Gin</td><td>Thr</td><td>5 ala</td><td>val</td><td>Tyr</td><td>SC?</td><td>Gin</td><td>10 Arg</td><td>Arg</td><td>Cys</td><td>How much</td><td>Gin</td><td>15 ala</td><td>Arg</td>

<td>Cheese</td><td>phe</td><td>Leu</td><td>twenty Pro</td><td>Gly</td><td>Leu</td><td>ala</td><td>Cheese</td><td>25 Arg</td><td>Thr</td><td>ala</td><td>Gly</td><td>Thr</td><td>thirty Pro</td><td>Cheese</td><td>ala</td>

<td>Thr</td><td>ala</td><td>35 Gin</td><td>Shaft</td><td>Thr</td><td colspan="2">Gly gly</td><td>40 own</td><td>lys</td><td>Gin</td><td>His</td><td>val</td><td>45 Kis</td><td>Pro</td><td>His</td><td>Gly</td>

<td>own</td><td>50 Cheese</td><td>val</td><td>own</td><td>Cheese</td><td>Gly</td><td>SS His</td><td>Glu</td><td>Glu</td><td>His</td><td>own</td><td>60 Thr</td><td>Arg</td><td>Trp</td><td>Pro</td><td>Arg</td>

<td>65 lys</td><td>val</td><td>phe</td><td>Cheese</td><td>Gly</td><td>70 How much</td><td>Gin</td><td>Pro</td><td>Thr</td><td>Gly</td><td>75 Cheese</td><td>Leu</td><td>His</td><td>Leu</td><td>Gly</td><td>80 own</td>

<td>Tyr</td><td>Leu</td><td>Gly</td><td>ala</td><td>85 val</td><td>Arg</td><td>lys</td><td>Trp</td><td>Shaft</td><td>90 Gin</td><td>Leu</td><td>Gin</td><td>own</td><td>ala</td><td>95 Arg</td><td>Asp</td>

<td>Asp</td><td>val</td><td>Thr</td><td>100 val</td><td>Cys</td><td>How much</td><td>val</td><td>Asp</td><td>105 Leu</td><td>His</td><td>Cheese</td><td>How much</td><td>Thr</td><td>110 Underworld</td><td>Pro</td><td>His</td>

<td>own</td><td>Pro</td><td>115 Pro</td><td>Leu</td><td>Leu</td><td>Arg</td><td>GILJ</td><td>120 own</td><td>how much</td><td>phe</td><td>Thr</td><td>mec</td><td>125 ala</td><td>ala</td><td>Thr</td><td>Leu</td>

<td>Leu</td><td>130 ala</td><td>Cys</td><td>Gly</td><td>How much</td><td>Asp</td><td>135 Pro</td><td>Thr</td><td>lys</td><td>Cheese</td><td>Thr</td><td>140 Leu</td><td>phe</td><td>Shaft</td><td>Gin</td><td>Cheese</td>

<td>145 ala</td><td>shaft</td><td>ala</td><td>Glu</td><td>His</td><td>150 ala</td><td>Glu</td><td>phe</td><td>own</td><td>Trp</td><td>155 How much</td><td>Leu</td><td>Cheese</td><td>Cheese</td><td>Leu</td><td>160 Thr</td>

<td>Thr</td><td>Underworld</td><td>Pro</td><td>Arg</td><td>165 Leu</td><td>ala</td><td>Gin</td><td>Leu</td><td>Pro</td><td>170 Gin</td><td>phe</td><td>Arg</td><td>Glu</td><td>lys</td><td>175 Cheese</td><td>Arg</td>

<td>Leu</td><td>Leu</td><td>lys</td><td>180 Asp</td><td>val</td><td>pro</td><td>Leu</td><td>Gly</td><td>185 Leu</td><td>Tyr</td><td>shaft</td><td>Tyr</td><td>Pro</td><td>190 shaft</td><td>Leu</td><td>Gin</td>

<td>ala</td><td>ala</td><td>195 Asp</td><td>How much</td><td>Underworld</td><td>Leu</td><td>Tyr</td><td>200 lys</td><td>Cheese</td><td>Thr</td><td>His</td><td>Shaft</td><td>20S Pro</td><td>Shaft</td><td>Gly</td><td>ala</td>

<td>Asp</td><td>210 Gin</td><td>How much</td><td>Gin</td><td>His</td><td>How much</td><td>215 Gin</td><td>Leu</td><td>ala</td><td>Gin</td><td>His</td><td>220 Leu</td><td>ala</td><td>Arg</td><td>How much</td><td>Tyr</td>

<td>225 own</td><td>Gly</td><td>Arg</td><td>Tyr</td><td>Gly</td><td>230 Glu</td><td>Thr</td><td>phe</td><td>Pro</td><td>val</td><td>235 Cys</td><td>Thr</td><td>ala</td><td>How much</td><td>How much</td><td>240 Glu</td>

<td>Asp</td><td>Gly</td><td>Asp</td><td>ala</td><td>24 S. cheese</td><td>Arg</td><td>vai</td><td>Leu</td><td>Cheese</td><td>250 Leu</td><td>Arg</td><td>Asp</td><td>Pro</td><td>Cheese</td><td>255 lys</td><td>lys</td>

<td>Underworld</td><td>Cheese</td><td>lys</td><td>260 Cheese</td><td>Glu</td><td>ala</td><td>own</td><td>Pro</td><td>265 lys</td><td>ala</td><td>Thr</td><td>How much</td><td>own</td><td>270 Leu</td><td>Cys</td><td>ASp</td>

<td>Cheese</td><td>Pro</td><td>275 Asp</td><td>Leu</td><td>How much</td><td>Thr</td><td>Gin</td><td>280 lys</td><td>How much</td><td>lys</td><td>lys</td><td>ala</td><td>285 Shaft</td><td>Thr</td><td>Asp</td><td>phe</td>

<td>Thr</td><td>290 Cheese</td><td>Asp</td><td>how much</td><td>Thr</td><td>Tyr</td><td>295 own</td><td>Pro</td><td>Gly</td><td>lys</td><td>Arg</td><td>300 ala</td><td>Gly</td><td>val</td><td>Cheese</td><td>own</td>

<td>.305 Leu</td><td>val</td><td>own</td><td>How much</td><td>His</td><td>310 ala</td><td>Gin</td><td>Shaft</td><td>Thr</td><td>Gly</td><td>315 Gin</td><td>Cheese</td><td>How much</td><td>lys</td><td>Thr</td><td>320 val</td>

<td>val</td><td>own</td><td>Glu</td><td>ala</td><td>325 ala</td><td>Thr</td><td>Leu</td><td>Asp</td><td>Thr</td><td>330 ala</td><td>lys</td><td>Tyr</td><td>lys</td><td>Asp</td><td>335 Arg</td><td>shaft</td>

<td>ala</td><td>Glu</td><td>ala</td><td>340 shaft</td><td>val</td><td>Glu</td><td>Kis</td><td>Leu</td><td>345 Arg</td><td>Pro</td><td>How much</td><td>Arg</td><td>Glu</td><td>350 Gin</td><td>How much</td><td>His</td>

<td>His</td><td>His</td><td>355 Underworld</td><td>Thr</td><td>lys</td><td>Arg</td><td>own</td><td>360 Glu</td><td>Underworld</td><td>How much</td><td>Tyr</td><td>Leu</td><td>365 Leu</td><td>Glu</td><td>Shaft</td><td>Gly</td>

<td>ala</td><td>370 Glu</td><td>lys</td><td>ala</td><td>Arg</td><td>Gin</td><td>375 Gin</td><td>ala</td><td>Arg</td><td>Gin</td><td>Thr</td><td>380 Leu</td><td>own</td><td>Asp</td><td>vai</td><td>lys</td>

<td>385 Gin</td><td>Arg</td><td>Leu</td><td>Gly</td><td>Leu</td><td>390 Gly</td><td>Thr</td><td>Cheese</td><td>ala</td><td>own</td><td>395 How much</td><td>Pro</td><td>ala</td><td>ala</td><td>val</td><td>400 His</td>

<td>shaft</td><td>ala</td><td>Pro</td><td>Leu</td><td>405 Leu</td><td>Pro</td><td>ala</td><td>Pro</td><td>Asp</td><td>410 how much</td><td>Cheese</td><td>lys</td><td>Thr</td><td>Cheese</td><td>415 ala</td><td>Cheese</td>

<td>Arg</td><td>Arg</td><td>Underworld</td><td>420 Cheese</td><td>lys</td><td>Asp</td><td>phe</td><td>Asp</td><td>425 Gly</td><td>own</td><td>val</td><td>His</td><td>Glu</td><td>430 Arg</td><td>His</td><td>Glu</td>

<td>Arg</td><td>Underworld</td><td>435 Tyr</td><td>aly</td><td>phe</td><td>Gly</td><td>Asp</td><td>440 Gin</td><td>Pro</td><td>Cheese</td><td>Gly</td><td>Gly</td><td>445 ala</td><td>Gly</td><td>Gin</td><td>Cheese</td>

<td>ala</td><td>450 Thr</td><td>Gly</td><td>Glu</td><td>Shaft</td><td>ala</td><td>455 Asp</td><td>Underworld</td><td>Pro</td><td>Gin</td><td>ala</td><td>460 val</td><td>val</td><td>Pro</td><td>Arg</td><td>Shaft</td>

-132

<td>46S</td>

<td>Ile Arg Arg Ala Pro</td>

<td> 485</td>

<td>Gin Leu Gly Ser Thr</td>

<td> 500</td>

<td>Asn Ala Pro His Ile</td>

<td> 515</td>

<td>Met val Thr Gly Ala</td>

<td> 530</td>

<td>Val Thr Gly Asn Leu</td>

<td> 545</td>

<td>Lys His Ser Gin Asn</td>

<td> 565</td>

<td>Glu Val Asn Ala Ala</td>

<td> 580</td>

<td>Tyr Gly Cheese Ala Leu</td>

<td> 595</td>

<td>Lys Thr Asn Ser Thr</td>

<td> 610</td>

<td>Glu Glu Glu Glu Gin</td>

<td> 625</td>

<td>Glu Glu Glu Glu Cheese</td>

<td> 645</td>

<td>Ala Thr Glu Ala Thr</td>

<td> 660</td>

<td colspan="5"> 470</td>

<td>How much</td><td>val</td><td>Pro</td><td>Thr</td><td>Gin</td>

<td>Arg</td><td>Thr</td><td>Arg</td><td>Hia</td><td>4 90 val</td>

<td>Gly</td><td>phe</td><td>lys</td><td>505 Pro</td><td>Pro</td>

<td>Leu</td><td>ala</td><td>520 lys</td><td>own</td><td>lys</td>

<td>Gly</td><td>535 phe</td><td>Gly</td><td>Gin</td><td>Gin</td>

<td>550 val</td><td>own</td><td>Pro</td><td>cheese</td><td>ala</td>

<td>lys</td><td>lys</td><td>Gin</td><td>Glu</td><td>570 phe</td>

<td>Glu</td><td>Cheese</td><td>Leu</td><td>585 Cheese</td><td>own</td>

<td>ala</td><td>Gly</td><td>600 Asp</td><td>Cheese</td><td>Glu</td>

<td>ala</td><td>615 ala</td><td>Cheese</td><td>Cheese</td><td>Glu</td>

<td>630 Gly</td><td>Glu</td><td>Arg</td><td>Glu</td><td>Arg</td>

<td>lys</td><td>val</td><td>Glu</td><td>how much 665</td><td> 650</td>

<td> 475</td><td></td><td></td><td></td><td></td><td> 480</td>

<td>Cheese</td><td>Underworld</td><td>Arg</td><td>val</td><td>Glu 4 95</td><td>lys</td>

<td>phe</td><td>Gin</td><td>Underworld</td><td>Asp 510</td><td>Thr</td><td>Cys</td>

<td>Pro</td><td>Gly</td><td>phe 525</td><td>Cheese</td><td>ala</td><td>Cheese</td>

<td>Arg</td><td>ala 540</td><td>Thr</td><td>val</td><td>lys</td><td>Pro</td>

<td>Cheese 555</td><td>Gin</td><td>Gly</td><td>Gly</td><td>Cheese</td><td>Tyr 560</td>

<td>val</td><td>Cheese</td><td>Thr</td><td>How much</td><td>ala 575</td><td>Arg</td>

<td>Thr</td><td>Cys</td><td>own</td><td>Pro 590</td><td>ala</td><td>Leu</td>

<td>Cheese</td><td>Cheese</td><td>Arg 605</td><td>Cheese</td><td>Cheese</td><td>Glu</td>

<td>phe</td><td>How much 620</td><td>How much</td><td>val</td><td>ala</td><td>Cheese</td>

<td>Glu 635</td><td>Gin</td><td>Arg</td><td>Glu</td><td>Glu</td><td>ala 640</td>

<td>Glu</td><td>lys</td><td>Gly</td><td>Glu</td><td>Arg 655</td><td>own</td>

&lt;210&gt; 63 &lt;211&gt; 360 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 63

<td>Underworld 1</td><td>ala</td><td>Leu</td><td>His</td><td>Cheese 5</td><td>Underworld</td><td>Arg</td><td>lys</td><td>ala</td><td>Arg 10</td><td>Glu</td><td>Arg</td><td>Trp</td><td>Cheese</td><td>phe 15</td><td>How much</td>

<td>Arg</td><td>ala</td><td>Leu</td><td>His twenty</td><td>lys</td><td>Gly</td><td>Cheese</td><td>ala</td><td>ala 25</td><td>ala</td><td>Pro</td><td>ala</td><td>Leu</td><td>Gin thirty</td><td>lys</td><td>Asp</td>

<td>Cheese</td><td>Lya</td><td>lys 35</td><td>Arg</td><td>val</td><td>phe</td><td>Cheese</td><td>Gly 40</td><td>how much</td><td>Gin</td><td>Pro</td><td>Thr</td><td>Gly 45</td><td>How much</td><td>Leu</td><td>His</td>

<td>Leu</td><td>Gly 50</td><td>own</td><td>Tyr</td><td>Leu</td><td>Gly</td><td>ala 55</td><td>How much</td><td>Glu</td><td>Cheese</td><td>Trp</td><td>val 60</td><td>Arg</td><td>Leu</td><td>Gin</td><td>Asp</td>

<td>Glu 65</td><td>Tyr</td><td>Asp</td><td>Cheese</td><td>vai</td><td>Leu 70</td><td>Tyr</td><td>cheese</td><td>How much</td><td>val</td><td>Asp 75</td><td>Leu</td><td>His</td><td>Cheese</td><td>How much</td><td>Thr 80</td>

<td>val</td><td>Pro</td><td>Gin</td><td>Asp</td><td>Pro B5</td><td>ala</td><td>val</td><td>Leu</td><td>Arg</td><td>Gin 90</td><td>Cheese</td><td>How much</td><td>Leu</td><td>Asp</td><td>Underworld 95</td><td>Thr</td>

<td>ala</td><td>val</td><td>Leu</td><td>Leu 100</td><td>ala</td><td>Cys</td><td>Gly</td><td>How much</td><td>own 105</td><td>Pro</td><td>Glu</td><td>lys</td><td>Cheese</td><td>How much 110</td><td>Leu</td><td>phe</td>

<td>Gin</td><td>Gin</td><td>Cheese 115</td><td>Gin</td><td>val</td><td>Cheese</td><td>Glu</td><td>His 120</td><td>Thr</td><td>Gin</td><td>Leu</td><td>cheese</td><td>Trp 125</td><td>how much</td><td>Leu</td><td>Cheese</td>

<td>Cys</td><td>Underworld 130</td><td>val</td><td>Arg</td><td>Leu</td><td>Pro</td><td>Arg 135</td><td>Leu</td><td>Gin</td><td>His</td><td>Leu</td><td>Kie 140</td><td>Gin</td><td>Trp</td><td>lys</td><td>ala</td>

<td>lys 145</td><td>Thr</td><td>Thr</td><td>lys</td><td>Gin</td><td>lys 150</td><td>His</td><td>Asp</td><td>Gly</td><td>Thr</td><td>val 155</td><td>Gly</td><td>Leu</td><td>Leu</td><td>Thr</td><td>Tyr 160</td>

<td>Pro</td><td>val</td><td>Leu</td><td>Gin</td><td>ala 165</td><td>ala</td><td>Asp</td><td>How much</td><td>Leu</td><td>Leu 170</td><td>Tyr</td><td>lys</td><td>Cheese</td><td>Thr</td><td>His 175</td><td>val</td>

<td>Pro</td><td>val</td><td>Gly</td><td>Glu iao</td><td>Asp</td><td>Gin</td><td>val</td><td>Gin</td><td>Hia 185</td><td>Underworld</td><td>Glu</td><td>Leu</td><td>val</td><td>Gin 190</td><td>aap</td><td>Leu</td>

<td>ala</td><td>Gin</td><td>Gly</td><td>phe</td><td>own</td><td>lys</td><td>lys</td><td>Tyr</td><td>Gly</td><td>Glu</td><td>phe</td><td>phe</td><td>Pro</td><td>val</td><td>Pro</td><td>Glu</td>

-133195 200 205

<td colspan="7">Cheese Ile Leu Thr Cheese Met Lys</td><td colspan="5" rowspan="2">Lys Val Lys Cheese Leu 220</td><td colspan="3" rowspan="2">Arg A3p Pro</td><td rowspan="2">Cheese</td>

<td colspan="6"> 210</td><td> 215</td>

<td>ala</td><td>lys</td><td>Underworld</td><td>Cheese</td><td>lys</td><td>Cheese</td><td>Asp</td><td>Pro</td><td>Asp</td><td>lys</td><td>Leu</td><td>ala</td><td>Thr</td><td>val</td><td>Arg</td><td>How much</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>Thr</td><td>Asp</td><td>Cheese</td><td>Pro</td><td>Glu</td><td>Glu</td><td>How much</td><td>val</td><td>Gin</td><td>lys</td><td>phe</td><td>Arg</td><td>lys</td><td>ala</td><td>val</td><td>Thr</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

<td>Asp</td><td>phe</td><td>Thr</td><td>Cheese</td><td>Glu</td><td>val</td><td>Thr</td><td>Tyr</td><td>Asp</td><td>Pro</td><td>ala</td><td>Gly</td><td>Arg</td><td>ala</td><td>Gly</td><td>val</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>Cheese</td><td>own</td><td>How much</td><td>val</td><td>ala</td><td>val</td><td>His</td><td>ala</td><td>ala</td><td>val</td><td>Thr</td><td>Gly</td><td>Leu</td><td>Cheese</td><td>val</td><td>Glu</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>Glu</td><td>val</td><td>val</td><td>Arg</td><td>Arg</td><td>Cheese</td><td>ala</td><td>Gly</td><td>Underworld</td><td>own</td><td>Thr</td><td>ala</td><td>Arg</td><td>Tyr</td><td>lys</td><td>Leu</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>ala</td><td>val</td><td>ala</td><td>Asp</td><td>ala</td><td>val</td><td>How much</td><td>Glu</td><td>lys</td><td>phe</td><td>ala</td><td>Pro</td><td>How much</td><td>Ly9</td><td>Arg</td><td>Glu</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

<td>how much</td><td>Glu</td><td>lys</td><td>Leu</td><td>lys</td><td>Leu</td><td>Asp</td><td>lys</td><td>Asp</td><td>His</td><td>Leu</td><td>Glu</td><td>lys</td><td>val</td><td>Leu</td><td>Gin</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td>

<td>How much</td><td>Gly</td><td>Cheese</td><td>ala</td><td>lys</td><td>ala</td><td>lys</td><td>Glu</td><td>Leu</td><td>ala</td><td>Tyr</td><td>Thr</td><td>val</td><td>Cys</td><td>Gin</td><td>Glu</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 34 5</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td>

<td>val</td><td>lys</td><td>lys</td><td>Leu</td><td>val</td><td>Gly</td><td>phe</td><td>Leu</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

355 360 &lt;210&gt; 64 &lt;211&gt; 379 &lt;212&gt; PRT &lt;213&gt; Saccharomyces cerevisiae &lt;400&gt; 64

<td>Underworld 1</td><td>Cheese</td><td>own</td><td>lys</td><td>Gin 5</td><td>ala</td><td>val</td><td>Leu</td><td>lys</td><td>Leu 10</td><td>How much</td><td>Cheese</td><td>lys</td><td>Arg</td><td>Trp 15</td><td>How much</td>

<td>Cheese</td><td>Thr</td><td>val</td><td>Gin twenty</td><td>Arg</td><td>ala</td><td>Asp</td><td>phe</td><td>lys 25</td><td>Leu</td><td>own</td><td>Cheese</td><td>Glu</td><td>ala thirty</td><td>Leu</td><td>His</td>

<td>Cheese</td><td>own</td><td>ala 35</td><td>Thr</td><td>val</td><td>phe</td><td>Cheese</td><td>Underworld 40</td><td>How much</td><td>Gin</td><td>Pro</td><td>Thr</td><td>Gly 45</td><td>Cys</td><td>phe</td><td>His</td>

<td>Leu</td><td>Gly 50</td><td>own</td><td>Tyr</td><td>Leu</td><td>Gly</td><td>ala 55</td><td>Thr</td><td>Arg</td><td>val</td><td>Trp</td><td>Thr 60</td><td>Asp</td><td>Leu</td><td>Cys</td><td>Glu</td>

<td>Leu € 5</td><td>lys</td><td>Gin</td><td>Pro</td><td>Gly</td><td>Gin 70</td><td>Glu</td><td>Leu</td><td>How much</td><td>phe</td><td>Gly 75</td><td>val</td><td>ala</td><td>Asp</td><td>Leu</td><td>His 80</td>

<td>ala</td><td>How much</td><td>Thr</td><td>val</td><td>Pro 85</td><td>lys</td><td>Pro</td><td>Asp</td><td>Gly</td><td>Glu 90</td><td>Underworld</td><td>phe</td><td>Arg</td><td>lys</td><td>phe 95</td><td>Arg</td>

<td>His</td><td>Glu</td><td>ala</td><td>val 100</td><td>ala</td><td>Cheese</td><td>How much</td><td>Leu</td><td>ala 105</td><td>val</td><td>Gly</td><td>val</td><td>Asp</td><td>Pro 110</td><td>Glu</td><td>lys</td>

<td>ala</td><td>Cheese</td><td>val 115</td><td>How much</td><td>Tyr</td><td>Gin</td><td>Cheese</td><td>Ala 12 0</td><td>How much</td><td>Pro</td><td>Gin</td><td>His</td><td>Cheese 125</td><td>Glu</td><td>Leu</td><td>His</td>

<td>Trp</td><td>Leu 130</td><td>Leu</td><td>Cheese</td><td>Thr</td><td>Leu</td><td>ala 135</td><td>Cheese</td><td>Underworld</td><td>Gly</td><td>Leu</td><td>Leu 140</td><td>own</td><td>Arg</td><td>Underworld</td><td>Thr</td>

<td>Gin 14S</td><td>Trp</td><td>Lye</td><td>Cheese</td><td>lys</td><td>Cheese 150</td><td>own</td><td>How much</td><td>lys</td><td>Gin</td><td>Cheese 155</td><td>Thr</td><td>own</td><td>Gly</td><td>Asp</td><td>Tyr 160</td>

<td>Leu</td><td>val</td><td>own</td><td>Asp</td><td>Cheese 165</td><td>Asp</td><td>val</td><td>Gly</td><td>lys</td><td>val 17 0</td><td>Arg</td><td>Leu</td><td>Gly</td><td>Leu</td><td>phe 175</td><td>Cheese</td>

<td>Tyr</td><td>Pro</td><td>val</td><td>Leu 180</td><td>Gin</td><td>ala</td><td>ala</td><td>Asp</td><td>How much 185</td><td>Leu</td><td>Leu</td><td>Tyr</td><td>lys</td><td>Cheese 190</td><td>Thr</td><td>His</td>

<td>vai</td><td>Pro</td><td>val 19S</td><td>Gly</td><td>Asp</td><td>Asp</td><td>Gin</td><td>Cheese 200</td><td>Gin</td><td>His</td><td>Leu</td><td>Glu</td><td>Leu 205</td><td>Thr</td><td>Arg</td><td>His</td>

<td>Leu</td><td>ala 210</td><td>Glu</td><td>lys</td><td>phe</td><td>own</td><td>lys 215</td><td>Underworld</td><td>Tyr</td><td>lys</td><td>lys</td><td>own 220</td><td>phe</td><td>phe</td><td>Pro</td><td>lys</td>

<td>Pro</td><td>val</td><td>Thr</td><td>Underworld</td><td>Leu</td><td>ala</td><td>Gin</td><td>Thr</td><td>lys</td><td>lys</td><td>val</td><td>Leu</td><td>cheese</td><td>Leu</td><td>Cheese</td><td>Thr</td>

-134225 230 235 240

Pro Glu Lys Lys Met Ser Lys Ser Asp Pro Asn His Asp Ser val Ile

245 250 255

Phe Leu Asn Asp Glu Pro Lys Ala Ile Gin Lys Lye Ile Arg Lys Ala

260 265 270

Leu Thr Asp Cheese Ile Ser Asp Arg Phe Tyr Tyr Asp Pro Val Glu Arg

275 280 285

Pro Gly Val Ser Asn Leu ile A3n Ile Val ser Gly ile Gin Arg Lys

290 295 300

Cheese ile Glu Asp val Val Glu Asp Val Cheese Arg Phe Asn Asn Tyr Arg

305 310 315 320

Asp Phe Lys Asp Tyr Val Cheese Glu Val Ile Ile Glu Glu Leu Lys Gly

325 330 335

Pro Arg Thr Glu Phe Glu Lys Tyr Ile Asn Glu Pro Thr Tyr Leu His

340 345 350

Cheese val val Glu Cheese Gly Met Arg Lys Ala Arg Glu Lys Ala Ala Lys

355 360 365

Asn Leu Ala Asp Ile His Lys Ile Met Gly Phe

370 375 &lt;210&gt; 65 &lt;211&gt; 475 &lt;212&gt; PRT &lt;213&gt; Xenopus laevis &lt;400&gt; 65

<td>Underworld 1</td><td>Cheese</td><td>Asp</td><td>Cheese</td><td>Gly 5</td><td>Thr</td><td>Cheese</td><td>Thr</td><td>Cheese</td><td>Gly 10</td><td>Cheese</td><td>Leu</td><td>Leu</td><td>Pro</td><td>Underworld 15</td><td>Asp</td>

<td>Leu</td><td>Tyr</td><td>own</td><td>Gin twenty</td><td>val</td><td>Thr</td><td>ala</td><td>Gin</td><td>Gly 25</td><td>Asp</td><td>lys</td><td>How much</td><td>Arg</td><td>val thirty</td><td>Leu</td><td>lys</td>

<td>Cheese</td><td>Glu</td><td>lys 35</td><td>Cheese</td><td>Pro</td><td>Lys</td><td>Glu</td><td>Glu 40</td><td>Ile</td><td>Asp</td><td>Ala</td><td>Ala</td><td>Val 45</td><td>Ly3</td><td>Leu</td><td>Leu</td>

<td>Leu</td><td>Ala 50</td><td>Leu</td><td>Lys</td><td>Val</td><td>Asp</td><td>Tyr 55</td><td>Lys</td><td>Asn</td><td>Val</td><td>Thr</td><td>Gly 60</td><td>Gin</td><td>Asp</td><td>Tyr</td><td>Lys</td>

<td>Pro 65</td><td>Gly</td><td>val</td><td>Pro</td><td>Pro</td><td>Ala 70</td><td>Asp</td><td>Asp</td><td>Met</td><td>Pro</td><td>Thr 75</td><td>Asn</td><td>Arg</td><td>Asn</td><td>Gly</td><td>pro 80</td>

<td>Ser</td><td>Thr</td><td>Pro</td><td>Asn</td><td>Asp 85</td><td>Gly</td><td>Asp</td><td>Asp</td><td>Phe</td><td>Val 90</td><td>Asp</td><td>Pro</td><td>Trp</td><td>Thr</td><td>Val 95</td><td>Gin</td>

<td>Thr</td><td>Gly</td><td>Ser</td><td>Ala 100</td><td>Lys</td><td>Gly</td><td>Val</td><td>Asp</td><td>Tyr 105</td><td>Asp</td><td>Lys</td><td>Leu</td><td>Ile</td><td>Val 110</td><td>Arg</td><td>phe</td>

<td>Gly</td><td>Ser</td><td>Ser 115</td><td>Lys</td><td>ile</td><td>Asp</td><td>Gin</td><td>Ala 120</td><td>Leu</td><td>Ile</td><td>Asp</td><td>Arg</td><td>Ile 125</td><td>Glu</td><td>Arg</td><td>Val</td>

<td>Thr</td><td>Gly 130</td><td>Gin</td><td>Lys</td><td>Ala</td><td>Hi3</td><td>His 13S</td><td>Phe</td><td>Leu</td><td>Arg</td><td>Arg</td><td>Gly 14 0</td><td>Ile</td><td>Phe</td><td>Phe</td><td>Ser</td>

<td>His 145</td><td>Arg</td><td>Asp</td><td>Met</td><td>His</td><td>Gin 150</td><td>Val</td><td>Leu</td><td>Asp</td><td>Ala</td><td>Tyr 1SS</td><td>Glu</td><td>Lys</td><td>Lys</td><td>Lys</td><td>Pro 160</td>

<td>Phe</td><td>Tyr</td><td>Leu</td><td>Tyr</td><td>Thr 165</td><td>Gly</td><td>Arg</td><td>Gly</td><td>Pro</td><td>Ser 170</td><td>Ser</td><td>Glu</td><td>Ala</td><td>Met</td><td>His 175</td><td>Val</td>

<td>Gly</td><td>His</td><td>Leu</td><td>Ile 180</td><td>Pro</td><td>Phe</td><td>Ile</td><td>Phe</td><td>Thr 185</td><td>Lys</td><td>Trp</td><td>Leu</td><td>Gin</td><td>Glu 190</td><td>Val</td><td>Phe</td>

<td>Asn</td><td>Val</td><td>Pro 195</td><td>Leu</td><td>Val</td><td>Val</td><td>Gin</td><td>Leu 200</td><td>Thr</td><td>Asp</td><td>Asp</td><td>Glu</td><td>Lys 205</td><td>Tyr</td><td>Leu</td><td>Trp</td>

<td>Lys</td><td>Asp 210</td><td>Leu</td><td>Thr</td><td>Leu</td><td>Glu</td><td>Lys 215</td><td>Ala</td><td>Tyr</td><td>Gin</td><td>Tyr</td><td>Ala 220</td><td>Thr</td><td>Glu</td><td>Asn</td><td>Ala</td>

<td>Lys 225</td><td>Asp</td><td>Ile</td><td>Ile</td><td>Ala</td><td>Cys 230</td><td>Gly</td><td>Phe</td><td>Asp</td><td>Val</td><td>Asn 235</td><td>Lys</td><td>Thr</td><td>Phe</td><td>Ile</td><td>Phe 240</td>

<td>Ser</td><td>Asp</td><td>Leu</td><td>Glu</td><td>Tyr</td><td>Met</td><td>Gly</td><td>Lys</td><td>Ser</td><td>Ser</td><td>Gly</td><td>Phe</td><td>Tyr</td><td>Gin</td><td>Asn</td><td>Val</td>

-135-

<td colspan="3"></td><td colspan="2"> 245</td><td colspan="8"> 250</td><td colspan="3"> 255</td>

<td>val</td><td>Lys</td><td>Val</td><td>Gln</td><td>Lys</td><td>His</td><td>Val</td><td>Thr</td><td>Phe</td><td>Asn</td><td>Gln</td><td>val</td><td>Lys</td><td>Gly</td><td>Ile</td><td>Phe</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>Gly</td><td>Phe</td><td>Thr</td><td>Asp</td><td>Ser</td><td>Asp</td><td>Cys</td><td>Ile</td><td>Gly</td><td>Lys</td><td>Ile</td><td>Ser</td><td>Phe</td><td>Pro</td><td>Ala</td><td>Ile</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>Gln</td><td>Ala</td><td>Ala</td><td>Pro</td><td>Ser</td><td>Phe</td><td>Ser</td><td>Ser</td><td>Ser</td><td>Phe</td><td>Pro</td><td>Glu</td><td>Ile</td><td>Phe</td><td>Lys</td><td>Gly</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>Arg</td><td>Lys</td><td>Asp</td><td>Ile</td><td>Gln</td><td>Cys</td><td>Leu</td><td>Ile</td><td>Pro</td><td>Cys</td><td>Ala</td><td>ile</td><td>Asp</td><td>Gln</td><td>Asp</td><td>Pro</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

<td>Tyr</td><td>Phe</td><td>Arg</td><td>Met</td><td>Thr</td><td>Arg</td><td>Asp</td><td>Val</td><td>Ala</td><td>Pro</td><td>Arg</td><td>Ile</td><td>Asn</td><td>Tyr</td><td>Pro</td><td>Lys</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td>

<td>Pro</td><td>Ala</td><td>Leu</td><td>Met</td><td>His</td><td>Ser</td><td>Thr</td><td>Phe</td><td>Phe</td><td>Pro</td><td>Ala</td><td>Leu</td><td>Gln</td><td>Gly</td><td>Ala</td><td>Gln</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td>

<td>Thr</td><td>Lys</td><td>Met</td><td>Ser</td><td>Ala</td><td>Ser</td><td>Asp</td><td>Pro</td><td>Asn</td><td>Ser</td><td>Ser</td><td>Ile</td><td>Phe</td><td>Leu</td><td>Thr</td><td>Asp</td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td>

<td>Thr</td><td>Ala</td><td>Lys</td><td>Gln</td><td>Ile</td><td>Lys</td><td>Ser</td><td>Lys</td><td>Ile</td><td>Asn</td><td>Lys</td><td>His</td><td>Ala</td><td>Phe</td><td>Ser</td><td>Gly</td>

<td></td><td> 370</td><td></td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>Lys</td><td>Asp</td><td>Thr</td><td>Ile</td><td>Glu</td><td>Glu</td><td>His</td><td>Arg</td><td>Gln</td><td>Phe</td><td>Gly</td><td>Gly</td><td>Asn</td><td>Trp</td><td>Glu</td>

<td> 385</td><td></td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td>

<td>vai</td><td>Asp</td><td>Val</td><td>Ser</td><td>Tyr</td><td>Met</td><td>Tyr</td><td>Leu</td><td>Thr</td><td>Phe</td><td>Phe</td><td>Leu</td><td>Glu</td><td>Asp</td><td>Asp</td><td>Glu</td>

<td></td><td></td><td></td><td></td><td> 405</td><td></td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td>

<td>Arg</td><td>Leu</td><td>Glu</td><td>Gln</td><td>ile</td><td>Lys</td><td>Gln</td><td>Asp</td><td>Tyr</td><td>ser</td><td>Ser</td><td>Gly</td><td>Ala</td><td>Leu</td><td>Leu</td><td>Thr</td>

<td></td><td></td><td></td><td> 420</td><td></td><td></td><td></td><td></td><td>42S</td><td></td><td></td><td></td><td></td><td> 430</td><td></td><td></td>

<td>Gly</td><td>Asp</td><td>Leu</td><td>Lys</td><td>Lys</td><td>Ile</td><td>Leu</td><td>Thr</td><td>Glu</td><td>Thr</td><td>Leu</td><td>Gln</td><td>Pro</td><td>Met</td><td>Ile</td><td>Ser</td>

<td></td><td></td><td> 435</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td></td><td></td><td></td>

<td>Ala</td><td>His</td><td>Gln</td><td>Glu</td><td>Arg</td><td>Arg</td><td>Lys</td><td>His</td><td>Ile</td><td>Thr</td><td>Glu</td><td>Glu</td><td>Thr</td><td>Val</td><td>Lys</td><td>Gln</td>

<td></td><td> 450</td><td></td><td></td><td></td><td></td><td> 455</td><td></td><td></td><td></td><td></td><td> 460</td><td></td><td></td><td></td><td></td>

<td>Phe</td><td>Met</td><td>Met</td><td>Pro</td><td>Arg</td><td>Lys</td><td>Leu</td><td>Ala</td><td>Phe</td><td>Asp</td><td>Phe</td><td></td><td></td><td></td><td></td><td></td>

<td> 465</td><td></td><td></td><td></td><td></td><td> 470</td><td></td><td></td><td></td><td></td><td> 475</td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt;66 &lt;211&gt; 417 &lt;212&gt; PRT &lt;213&gt; Caenorhabditis elegans &lt;400&gt; 66

<td>Met 1</td><td>Ala</td><td>Ala</td><td>Pro</td><td>Ala 5</td><td>Glu</td><td>Gln</td><td>Val</td><td>Ala</td><td>Ala 10</td><td>Glu</td><td>Ile</td><td>Glu</td><td>Asn</td><td>Leu 15</td><td>Lys</td>

<td>Val</td><td>Asn</td><td>Gly</td><td>Gly 20</td><td>Ala</td><td>Ala</td><td>Gly</td><td>Gly</td><td>Gly 25</td><td>Val</td><td>Gln</td><td>Glu</td><td>Asp</td><td>Glu 30</td><td>Glu</td><td>Asp</td>

<td>Arg</td><td>Val</td><td>Thr 35</td><td>Pro</td><td>Trp</td><td>Glu</td><td>Val</td><td>Thr 40</td><td>Thr</td><td>Thr</td><td>Lys</td><td>Ala</td><td>Thr 45</td><td>Gly</td><td>Ile</td><td>Aap</td>

<td>Tyr</td><td>Asp 50</td><td>Lys</td><td>Leu</td><td>ile</td><td>Val</td><td>Lys 55</td><td>Phe</td><td>Gly</td><td>Cys</td><td>Arg</td><td>Lys 60</td><td>Leu</td><td>Asp</td><td>GlU</td><td>Glu</td>

<td>Ile 6S</td><td>Ile</td><td>Ala</td><td>Arg</td><td>Phe</td><td>Glu 70</td><td>Arg</td><td>Val</td><td>Thr</td><td>Gly</td><td>His 75</td><td>Lys</td><td>Ala</td><td>Ser</td><td>Pro</td><td>Met 80</td>

<td>Leu</td><td>Arg</td><td>Arg</td><td>Gly</td><td>Met 85</td><td>Phe</td><td>Phe</td><td>Ala</td><td>His</td><td>Arg 90</td><td>Asp</td><td>Leu</td><td>Thr</td><td>Ala</td><td>Ile 95</td><td>Leu</td>

<td>Asp</td><td>Arg</td><td>Lys</td><td>Glu 100</td><td>Gln</td><td>Gly</td><td>Lys</td><td>Pro</td><td>Phe 105</td><td>Tyr</td><td>Leu</td><td>Tyr</td><td>Thr</td><td>Gly 110</td><td>Arg</td><td>Gly</td>

<td>Ala</td><td>Ser</td><td>Ser 115</td><td>Gly</td><td>Ser</td><td>Leu</td><td>His</td><td>Leu 120</td><td>Gly</td><td>His</td><td>Leu</td><td>val</td><td>Pro 125</td><td>Phe</td><td>Ile</td><td>Phe</td>

<td>Thr</td><td>Lys 130</td><td>Trp</td><td>Leu</td><td>Gln</td><td>Glu</td><td>Val 135</td><td>Phe</td><td>Asp</td><td>Val</td><td>Pro</td><td>Leu 140</td><td>Val</td><td>Ile</td><td>Gln</td><td>Met</td>

<td>Thr 145</td><td>Asp</td><td>ASp</td><td>Glu</td><td>Lys</td><td>Phe 150</td><td>Leu</td><td>Trp</td><td>Lys</td><td>Aap</td><td>Met 155</td><td>Lys</td><td>Val</td><td>Asp</td><td>Glu</td><td>Ala 160</td>

<td>Lys</td><td>Lys</td><td>Met</td><td>Ala</td><td>Arg</td><td>Glu</td><td>Asn</td><td>Met</td><td>Lys</td><td>Asp</td><td>Ile</td><td>Ile</td><td>Ser</td><td>Val</td><td>Gly</td><td>Phe</td>

-136-

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Asp</td><td>Pro</td><td>Thr</td><td>Lys 180</td><td>Thr</td><td>Phe</td><td>Ile</td><td>Phe</td><td>Asn 185</td><td>Aan</td><td>Phe</td><td>Asp</td><td>Tyr</td><td>Met 190</td><td>Cys</td><td>Pro</td>

<td>Pro</td><td>Phe</td><td>Tyr 195</td><td>GlU</td><td>Asn</td><td>Ile</td><td>val</td><td>Lya 200</td><td>Ile</td><td>Trp</td><td>Lys</td><td>Vał</td><td>Val 205</td><td>Asn</td><td>Thr</td><td>Asn</td>

<td>Gin</td><td>Ala 210</td><td>Arg</td><td>Ala</td><td>Ile</td><td>Phe</td><td>Gly 215</td><td>Phe</td><td>Thr</td><td>Pro</td><td>Glu</td><td>Asp 220</td><td>Cys</td><td>Leu</td><td>Gly</td><td>Lys</td>

<td>Ala 225</td><td>Ala</td><td>Phe</td><td>Pro</td><td>Ala</td><td>Val 230</td><td>Glu</td><td>Ala</td><td>Ala</td><td>Pro</td><td>Cys 235</td><td>Phe</td><td>Ala</td><td>Ser</td><td>Ser</td><td>Phe 240</td>

<td>Pro</td><td>Gin</td><td>Ile</td><td>Phe</td><td>Gly 245</td><td>Lys</td><td>Arg</td><td>Asn</td><td>Asp</td><td>Ile 250</td><td>Pro</td><td>Cys</td><td>Leu</td><td>Ile</td><td>Pro 255</td><td>cys</td>

<td>Ala</td><td>Ile</td><td>Asp</td><td>Gin 260</td><td>Asp</td><td>Pro</td><td>Phe</td><td>Phe</td><td>Arg 265</td><td>Met</td><td>Thr</td><td>Arg</td><td>Asp</td><td>Val 270</td><td>Ala</td><td>Pro</td>

<td>Arg</td><td>Leu</td><td>Lys 275</td><td>Ala</td><td>Ser</td><td>Lys</td><td>Pro</td><td>Ser 280</td><td>Leu</td><td>Ile</td><td>Phe</td><td>Ser</td><td>Thr 285</td><td>Phe</td><td>Leu</td><td>Pro</td>

<td>Ala</td><td>Leu 290</td><td>Thr</td><td>Gly</td><td>Ala</td><td>Gin</td><td>Thr 29S</td><td>Lys</td><td>Met</td><td>Ser</td><td>Ala</td><td>Ser 300</td><td>Glu</td><td>Pro</td><td>Asn</td><td>Thr</td>

<td>Cys 305</td><td>Ile</td><td>Phe</td><td>Leu</td><td>Ser</td><td>Asp 310</td><td>Thr</td><td>Ala</td><td>Lys</td><td>Gin</td><td>Ile 315</td><td>Lys</td><td>Asn</td><td>Lys</td><td>Ile</td><td>Asn 320</td>

<td>Lys</td><td>Tyr</td><td>Ala</td><td>Phe</td><td>Ser 325</td><td>Gly</td><td>Gly</td><td>Gin</td><td>Gin</td><td>Thr 330</td><td>Val</td><td>Sin</td><td>Glu</td><td>His</td><td>Arg 335</td><td>Glu</td>

<td>Lys</td><td>Gly</td><td>Gly</td><td>Asn 340</td><td>Cys</td><td>Asp</td><td>Val</td><td>Asp</td><td>Ile 345</td><td>Ser</td><td>Tyr</td><td>Gin</td><td>Phe</td><td>Leu 350</td><td>Arg</td><td>Phe</td>

<td>Phe</td><td>Leu</td><td>Asp 355</td><td>Asp</td><td>Asp</td><td>Glu</td><td>Lys</td><td>Leu 360</td><td>Ala</td><td>Glu</td><td>Ile</td><td>Arg</td><td>Glu 365</td><td>Asn</td><td>Tyr</td><td>Thr</td>

<td>Lys</td><td>Gly 370</td><td>Glu</td><td>Met</td><td>Leu</td><td>Ser</td><td>Gly 375</td><td>Glu</td><td>Leu</td><td>Lys</td><td>Ala</td><td>Leu 380</td><td>Ala</td><td>Thr</td><td>Gin</td><td>Lys</td>

<td>Val 385</td><td>Gin</td><td>Glu</td><td>Ile</td><td>Val</td><td>Leu 390</td><td>Glu</td><td>Met</td><td>Gin</td><td>Glu</td><td>Arg 395</td><td>Arg</td><td>Lys</td><td>Leu</td><td>val</td><td>Thr 400</td>

<td>Asp</td><td>Glu</td><td>Thr</td><td>Val</td><td>Glu 405</td><td>GlU</td><td>Phe</td><td>Val</td><td>Lys</td><td>Val 410</td><td>Arg</td><td>Pro</td><td>Leu</td><td>Ala</td><td>Tyr 415</td><td>Lys</td>

Tyr &lt;210&gt;67 &lt;211&gt; 337 &lt;212&gt; PRT &lt;213&gt; Salmonella enterica subsp. enterica serovar Typhi8 &lt;400&gt; 67

<td>Met 1</td><td>Asn</td><td>Thr</td><td>Leu</td><td>Pro 5</td><td>Ile</td><td>Ile</td><td>Leu</td><td>Thr</td><td>Gly 10</td><td>Asp</td><td>Arg</td><td>Pro</td><td>Thr</td><td>Gly 1S</td><td>ser</td>

<td>Leu</td><td>His</td><td>Leu</td><td>Gly 20</td><td>His</td><td>Tyr</td><td>Val</td><td>Gly</td><td>Ser 25</td><td>Leu</td><td>Arg</td><td>Gin</td><td>Arg</td><td>Val 30</td><td>Ala</td><td>Leu</td>

<td>Gin</td><td>Gin</td><td>Asp 35</td><td>His</td><td>Gin</td><td>Gin</td><td>Tyr</td><td>Val 40</td><td>Leu</td><td>Ile</td><td>Ala</td><td>Asp</td><td>Leu 45</td><td>Gin</td><td>Gly</td><td>Leu</td>

<td>Thr</td><td>Asp 50</td><td>Asn</td><td>Gly</td><td>Ser</td><td>Asn</td><td>Pro 55</td><td>Gin</td><td>Lys</td><td>Ile</td><td>Arg</td><td>Asp 60</td><td>Asn</td><td>ile</td><td>Pro</td><td>Gin</td>

<td>Val 65</td><td>Leu</td><td>Ala</td><td>ASp</td><td>Tyr</td><td>Leu 70</td><td>Ala</td><td>Val</td><td>Gly</td><td>Ile</td><td>Asp 75</td><td>Pro</td><td>Ala</td><td>Leu</td><td>Thr</td><td>Thr 80</td>

<td>Ile</td><td>Cys</td><td>Leu</td><td>Gin</td><td>Ser 85</td><td>Ala</td><td>Leu</td><td>Pro</td><td>Ala</td><td>Leu 90</td><td>Ala</td><td>Glu</td><td>Leu</td><td>Thr</td><td>Val 95</td><td>Leu</td>

<td>Tyr</td><td>Mec</td><td>Asn</td><td>Ile 100</td><td>val</td><td>Thr</td><td>val</td><td>Ala</td><td>Arg 105</td><td>Val</td><td>Glu</td><td>Arg</td><td>Asn</td><td>Pro 110</td><td>Thr</td><td>Val</td>

<td>Lys</td><td>Asn</td><td>Glu 115</td><td>Ile</td><td>Ala</td><td>Gin</td><td>Lys</td><td>Gly 120</td><td>Phe</td><td>Thr</td><td>Arg</td><td>ser</td><td>Leu 125</td><td>Pro,</td><td>Val</td><td>Gly</td>

<td>Phe</td><td>Met</td><td>Ala</td><td>Tyr</td><td>Pro</td><td>Ile</td><td>Ser</td><td>Gin</td><td>Ala</td><td>Ala</td><td>Asp</td><td>Ile</td><td>Thr</td><td>Ala</td><td>Phe</td><td>Lys</td>

-137-

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Ala 14 S</td><td>Glu</td><td>Met</td><td>Val</td><td>Pro</td><td>Val ISO</td><td>Gly</td><td>Asp</td><td>Asp</td><td>Gin</td><td>Leu 155</td><td>Pro</td><td>Met</td><td>Ile</td><td>Glu</td><td>Gin 160</td>

<td>Thr</td><td>Asn</td><td>Glu</td><td>ile</td><td>Val 165</td><td>His</td><td>Lys</td><td>Met</td><td>Asn</td><td>Ser 170</td><td>Leu</td><td>Phe</td><td>Ser</td><td>Ser</td><td>Pro 175</td><td>Val</td>

<td>Leu</td><td>Arg</td><td>Pro</td><td>Cys 130</td><td>Gin</td><td>Ala</td><td>Leu</td><td>Leu</td><td>Ser 185</td><td>Asp</td><td>Thr</td><td>Gly</td><td>Arg</td><td>Leu 190</td><td>Pro</td><td>Gly</td>

<td>Ile</td><td>Asp</td><td>Gly 195</td><td>Ser</td><td>Ala</td><td>Lys</td><td>Met</td><td>Ser 200</td><td>Lys</td><td>Ser</td><td>Leu</td><td>Val</td><td>Asn 205</td><td>Thr</td><td>Leu</td><td>Leu</td>

<td>Leu</td><td>Ser 210</td><td>Ala</td><td>Ser</td><td>Glu</td><td>Glu</td><td>Thr 215</td><td>Ile</td><td>His</td><td>Arg</td><td>Ala</td><td>Val 220</td><td>Ser</td><td>Ala</td><td>Met</td><td>Tyr</td>

<td>Thr 22S</td><td>Asp</td><td>Pro</td><td>Asn</td><td>His</td><td>Leu 230</td><td>Lys</td><td>ile</td><td>Ser</td><td>Asp</td><td>Pro 235</td><td>Gly</td><td>Lys</td><td>Ile</td><td>Glu</td><td>Gly 240</td>

<td>Asn</td><td>Val</td><td>Val</td><td>Phe</td><td>Thr 245</td><td>Trp</td><td>Leu</td><td>Asp</td><td>Ala</td><td>Phe 250</td><td>His</td><td>Pro</td><td>Asp</td><td>Lys</td><td>Ala 255</td><td>Lys</td>

<td>Val</td><td>Ala</td><td>Ala</td><td>Met 260</td><td>Lys</td><td>Ala</td><td>His</td><td>Tyr</td><td>Gin 265</td><td>Gin</td><td>Gly</td><td>Gly</td><td>Leu</td><td>Gly 270</td><td>Asp</td><td>Arg</td>

<td>Val</td><td>Cys</td><td>Lys 275</td><td>Asn</td><td>Glu</td><td>Leu</td><td>Glu</td><td>Thr 2S0</td><td>Cys</td><td>Leu</td><td>Gin</td><td>Glu</td><td>Leu 285</td><td>Ile</td><td>Ala</td><td>Pro</td>

<td>Ile</td><td>Arg 290</td><td>Glu</td><td>Arg</td><td>Arg</td><td>Ala</td><td>Thr 295</td><td>Phe</td><td>Ile</td><td>Ala</td><td>Asp</td><td>Lys 300</td><td>Gly</td><td>Met</td><td>Leu</td><td>Met</td>

<td>Glu 305</td><td>Leu</td><td>Leu</td><td>Lys</td><td>Lys</td><td>Gly 310</td><td>Ser</td><td>Glu</td><td>Arg</td><td>Ala</td><td>His 315</td><td>Glu</td><td>Val</td><td>Thr</td><td>Gin</td><td>Lys 320</td>

<td>Thr</td><td>Leu</td><td>Gin</td><td>Glu</td><td>Val 32S</td><td>Lys</td><td>Arg</td><td>Gly</td><td>Leu</td><td>Gly 330</td><td>Leu</td><td>Pro</td><td>Thr</td><td>Leu</td><td>Phe 335</td><td>Gin</td>

Val &lt;210&gt; 68 &lt;211&gt; 448 &lt;212&gt; PRT &lt;213&gt; Pseudomonas aeruginosa &lt;400&gt; 68

<td>Met</td><td>Thr</td><td>Thr</td><td>Arg</td><td>ile</td><td>Leu</td><td>Thr</td><td>Gly</td><td>Ile</td><td>Thr</td><td>Pro</td><td>Thr</td><td>Gly</td><td>Thr</td><td>Pro</td><td>His</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Leu</td><td>Gly</td><td>Asn</td><td>Tyr</td><td>Ala</td><td>Gly</td><td>Ala</td><td>Ile</td><td>Arg</td><td>Pro</td><td>Ala</td><td>Ile</td><td>Leu</td><td>Ala</td><td>ser</td><td>Arg</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Arg</td><td>Ser</td><td>Asp</td><td>Val</td><td>Asp</td><td>Ser</td><td>Phe</td><td>Tyr</td><td>Phe</td><td>Leu</td><td>Ala</td><td>Asp</td><td>Tyr</td><td>His</td><td>Ala</td><td>Leu</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Ile</td><td>Lys</td><td>Cys</td><td>Asp</td><td>Asp</td><td>Pro</td><td>Ala</td><td>Arg</td><td>Ile</td><td>Gin</td><td>Arg</td><td>Ser</td><td>Arg</td><td>Leu</td><td>Glu</td><td>Ile</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Ala</td><td>Ala</td><td>Thr</td><td>Trp</td><td>Leu</td><td>Ala</td><td>Gly</td><td>Gly</td><td>Leu</td><td>Asp</td><td>Val</td><td>Glu</td><td>Arg</td><td>Ala</td><td>Thr</td><td>Phe</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Tyr</td><td>Arg</td><td>Gin</td><td>Ser</td><td>Asp</td><td>Ile</td><td>Pro</td><td>Glu</td><td>Ile</td><td>Pro</td><td>Glu</td><td>Leu</td><td>Thr</td><td>trp</td><td>Leu</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Thr</td><td>Cy3</td><td>val</td><td>Ser</td><td>Ala</td><td>Lys</td><td>Gly</td><td>Leu</td><td>Leu</td><td>Asn</td><td>Arg</td><td>Ala</td><td>His</td><td>Ala</td><td>Tyr</td><td>Lys</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Ala</td><td>Ala</td><td>Val</td><td>Asp</td><td>Arg</td><td>Asn</td><td>Val</td><td>Glu</td><td>Ala</td><td>Gly</td><td>Glu</td><td>Asp</td><td>Pro</td><td>Asp</td><td>Ala</td><td>Gly</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Val</td><td>Thr</td><td>Met</td><td>Gly</td><td>Leu</td><td>Tyr</td><td>Ser</td><td>Tyr</td><td>Pro</td><td>Val</td><td>Leu</td><td>Met</td><td>Ala</td><td>Ala</td><td>Asp</td><td>Ile</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Leu</td><td>Met</td><td>Phe</td><td>Asn</td><td>Ala</td><td>His</td><td>Ly9</td><td>Ile</td><td>Pro</td><td>Val</td><td>Gly</td><td>Arg</td><td>Asp</td><td>Gin</td><td>Val</td><td>Gin</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Hia</td><td>Val</td><td>Glu</td><td>Met</td><td>Ala</td><td>Arg</td><td>Asp</td><td>ile</td><td>Gly</td><td>Gin</td><td>Arg</td><td>Phe</td><td>Asn</td><td>His</td><td>Leu</td><td>Phe</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Gly</td><td>Asn</td><td>Gly</td><td>Arg</td><td>Glu</td><td>Phe</td><td>Phe</td><td>Val</td><td>Leu</td><td>Pro</td><td>Glu</td><td>Ala</td><td>Val</td><td>Ile</td><td>Glu</td><td>Glu</td>

-138-

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Asn</td><td>Val</td><td>Ala 195</td><td>Thr</td><td>Leu</td><td>Pro</td><td>Gly</td><td>Lei' 200</td><td>'Asp</td><td>Gly</td><td>Arg</td><td>Lys</td><td>Met 20S</td><td>Ser</td><td>Lys</td><td>Ser</td>

<td>Tyr</td><td>Asp 210</td><td>Asn</td><td>Thr</td><td>Ile</td><td>Pro</td><td>Leu 215</td><td>Phe</td><td>Ser</td><td>Pro</td><td>Ser</td><td>Arg 220</td><td>Gin</td><td>Leu</td><td>Lye</td><td>Asp</td>

<td>Ala 22S</td><td>ile</td><td>Ala</td><td>Arg</td><td>Ile</td><td>Val 230</td><td>Thr</td><td>Asp</td><td>Ser</td><td>Arg</td><td>Ala 235</td><td>Pro</td><td>Gly</td><td>Glu</td><td>Pro</td><td>Lys 240</td>

<td>Asp</td><td>Pro</td><td>Asp</td><td>Ser</td><td>Ser 245</td><td>His</td><td>Leu</td><td>Phe</td><td>Leu</td><td>Leu 250</td><td>Tyr</td><td>Ser</td><td>Ala</td><td>Phe</td><td>Ala 255</td><td>Ser</td>

<td>Ala</td><td>Glu</td><td>Gin</td><td>Val 260</td><td>Ala</td><td>Ala</td><td>Phe</td><td>Arg</td><td>Gin 265</td><td>Glu</td><td>Leu</td><td>Leu</td><td>Glu</td><td>Gly 270</td><td>Leu</td><td>Ala</td>

<td>Trp</td><td>Gly</td><td>Glu 275</td><td>Ala</td><td>Lys</td><td>Gin</td><td>Arg</td><td>Leu 280</td><td>Phe</td><td>Gin</td><td>Leu</td><td>Leu</td><td>Asp 285</td><td>Asn</td><td>Glu</td><td>Leu</td>

<td>Gly</td><td>Glu 290</td><td>Ala</td><td>Arg</td><td>Glu</td><td>Arg</td><td>Tyr 2 95</td><td>Gin</td><td>Ala</td><td>Leu</td><td>Ile</td><td>Ala 300</td><td>Lys</td><td>Pro</td><td>Asp</td><td>Asp</td>

<td>Ile 305</td><td>Glu</td><td>Asp</td><td>Ile</td><td>Leu</td><td>Leu 310</td><td>Ala</td><td>Gly</td><td>Ala</td><td>Ala</td><td>Lys 315</td><td>Ala</td><td>Arg</td><td>Arg</td><td>ile</td><td>Ala 320</td>

<td>Thr</td><td>Pro</td><td>Phe</td><td>Ile</td><td>Ala 325</td><td>Glu</td><td>Leu</td><td>Arg</td><td>Glu</td><td>Ala 330</td><td>Val</td><td>Gly</td><td>Leu</td><td>Arg</td><td>Ser, 335</td><td>Leu</td>

<td>Arg</td><td>Glu</td><td>Pro</td><td>Leu 340</td><td>Lys</td><td>Ser</td><td>Ala</td><td>Glu</td><td>ser 345</td><td>Gly</td><td>Lys</td><td>Lys</td><td>Lys</td><td>Ala 350</td><td>Ala</td><td>Lys</td>

<td>Ala</td><td>Ala</td><td>Arg 355</td><td>Leu</td><td>Val</td><td>Ser</td><td>Phe</td><td>Arg 360</td><td>Asp</td><td>Asp</td><td>Asp</td><td>Gly</td><td>Ser 365</td><td>Phe</td><td>Arg</td><td>Phe</td>

<td>Arg</td><td>Leu 370</td><td>Leu</td><td>Asp</td><td>Ala</td><td>Ala</td><td>Gly 375</td><td>Glu</td><td>Gin</td><td>Leu</td><td>Leu</td><td>Leu 380</td><td>Ser</td><td>Arg</td><td>Ala</td><td>Phe</td>

<td>Ala 38S</td><td>Asp</td><td>Gly</td><td>Lys</td><td>Ala</td><td>Ala 390</td><td>Gly</td><td>Ala</td><td>val</td><td>Ser</td><td>Lys 3 95</td><td>Arg</td><td>Leu</td><td>Leu</td><td>Ala</td><td>Gly 400</td>

<td>Glu</td><td>Thr</td><td>Ala</td><td>Asp</td><td>Leu 405</td><td>Arg</td><td>Ala</td><td>Glu</td><td>Gly</td><td>Asn 410</td><td>Ala</td><td>Phe</td><td>Gly</td><td>Leu</td><td>Trp 415</td><td>Leu</td>

<td>Asp</td><td>Gly</td><td>Glu</td><td>Ala 420</td><td>Val</td><td>Ala</td><td>Gin</td><td>Ser</td><td>pro 425</td><td>Ala</td><td>Phe</td><td>Ala</td><td>Asp</td><td>Ala 430</td><td>Ala</td><td>Ala</td>

<td>Arg</td><td>Asp</td><td>Ala 435</td><td>Ala</td><td>Ile</td><td>Glu</td><td>Arg</td><td>Thr 440</td><td>Arg</td><td>Glu</td><td>Ala</td><td>Leu</td><td>Ala 445</td><td>Pro</td><td>Gin</td><td>Glu</td>

&lt;210&gt; 69 &lt;211&gt; 350 &lt;212&gt; PRT &lt;213&gt; Thermus thermophiles &lt;400&gt; 69

<td>Met 1</td><td>Leu</td><td>Glu</td><td>Glu</td><td>Ala s</td><td>Leu</td><td>Ala</td><td>Ala</td><td>Ile</td><td>Gin 10</td><td>Asn</td><td>Ala</td><td>Arg</td><td>Asp</td><td>Leu 15</td><td>Glu</td>

<td>Glu</td><td>Leu</td><td>Lys</td><td>Ala 20</td><td>Leu</td><td>Lys</td><td>Ala</td><td>Arg</td><td>Tyr 25</td><td>Leu</td><td>Gly</td><td>Lys</td><td>Lya</td><td>Gly 30</td><td>Leu</td><td>Leu</td>

<td>Thr</td><td>Gin</td><td>Glu 35</td><td>Met</td><td>Lys</td><td>Gly</td><td>Leu</td><td>Ser 40</td><td>Ala</td><td>Leu</td><td>Pro</td><td>Leu</td><td>Glu 45</td><td>Glu</td><td>Arg</td><td>Arg</td>

<td>Lys</td><td>Arg 50</td><td>Gly</td><td>Gin</td><td>Glu</td><td>Leu</td><td>Asn 55</td><td>Ala</td><td>Ile</td><td>Lys</td><td>Ala</td><td>Ala 60</td><td>Leu</td><td>Glu</td><td>Ala</td><td>Ala</td>

<td>Leu 65</td><td>Glu</td><td>Ala</td><td>Arg</td><td>Glu</td><td>Lys 70</td><td>Ala</td><td>Leu</td><td>Glu</td><td>Glu</td><td>Ala 75</td><td>Ala</td><td>Leu</td><td>Lys</td><td>Glu</td><td>Ala 80</td>

<td>Leu</td><td>Glu</td><td>Arg</td><td>Glu</td><td>Arg 85</td><td>Val</td><td>A3p</td><td>Val</td><td>Ser</td><td>Leu 90</td><td>Pro</td><td>Gly</td><td>Ala</td><td>Ser</td><td>Leu 95</td><td>Phe</td>

<td>Ser</td><td>Gly</td><td>Gly</td><td>Leu 100</td><td>His</td><td>Pro</td><td>Ile</td><td>Thr</td><td>Leu 105</td><td>Met</td><td>Glu</td><td>Arg</td><td>Glu</td><td>Leu 110</td><td>val</td><td>Glu</td>

<td>Ile</td><td>Phe</td><td>Arg 115</td><td>Ala</td><td>Leu</td><td>Gly</td><td>Tyr</td><td>Gin 120</td><td>Ala</td><td>Val</td><td>Glu</td><td>Gly</td><td>Pro 125</td><td>Glu</td><td>Val</td><td>Glu</td>

<td>ser</td><td>Glu</td><td>Phe</td><td>Phe</td><td>Asn</td><td>Phe</td><td>Asp</td><td>Ala</td><td>Leu</td><td>Asn</td><td>Ile</td><td>Pro</td><td>Glu</td><td>His</td><td>His</td><td>Pro</td>

-139130 135 140

<td>Ala</td><td>Arg</td><td>Asp</td><td>Met</td><td>Trp</td><td>Asp</td><td>Thr</td><td>Phe</td><td>Trp</td><td>Leu</td><td>Thr</td><td>Gly</td><td>Glu</td><td>Gly</td><td>Phe</td><td>Arg</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Leu</td><td>Glu</td><td>Gly</td><td>Pro</td><td>Leu</td><td>Gly</td><td>Glu</td><td>Glu</td><td>Val</td><td>Glu</td><td>Gly</td><td>Arg</td><td>Leu</td><td>Leu</td><td>Leu</td><td>Arg</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td>i7S</td><td></td>

<td>Thr</td><td>His</td><td>Thr</td><td>Ser</td><td>Pro</td><td>Met</td><td>Gin</td><td>Val</td><td>Arg</td><td>Tyr</td><td>Met</td><td>Val</td><td>Ala</td><td>His</td><td>Thr</td><td>Pro</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Pro</td><td>Phe</td><td>Arg</td><td>Ile</td><td>Val</td><td>Val</td><td>Pro</td><td>Gly</td><td>Arg</td><td>Val</td><td>Phe</td><td>Arg</td><td>Phe</td><td>Glu</td><td>Gin</td><td>Thr</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Asp</td><td>Ala</td><td>Thr</td><td>His</td><td>Glu</td><td>Ala</td><td>Val</td><td>Phe</td><td>His</td><td>Gin</td><td>Leu</td><td>Glu</td><td>Gly</td><td>Leu</td><td>Val</td><td>Val</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td>21S</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>Glu</td><td>Gly</td><td>Ile</td><td>Ala</td><td>Met</td><td>Ala</td><td>Hi3</td><td>Leu</td><td>Lys</td><td>Gly</td><td>Ala</td><td>Ile</td><td>Tyr</td><td>Glu</td><td>Leu</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 23 5</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>Ala</td><td>Gin</td><td>Ala</td><td>Leu</td><td>Phe</td><td>Gly</td><td>Pro</td><td>Asp</td><td>Ser</td><td>Lys</td><td>Val</td><td>Arg</td><td>Phe</td><td>Gin</td><td>Pro</td><td>Val</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

<td>Tyr</td><td>Phe</td><td>Pro</td><td>Phe</td><td>val</td><td>Glu</td><td>Pro</td><td>Gly</td><td>Ala</td><td>Gin</td><td>Phe</td><td>Ala</td><td>val</td><td>Trp</td><td>Trp</td><td>Pro</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>Glu</td><td>Gly</td><td>Gly</td><td>Lys</td><td>Trp</td><td>Leu</td><td>Glu</td><td>Leu</td><td>Gly</td><td>Gly</td><td>Ala</td><td>Gly</td><td>Met</td><td>Val</td><td>His</td><td>Pro</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>Lys</td><td>val</td><td>Phe</td><td>Gin</td><td>Ala</td><td>Val</td><td>Asp</td><td>Ala</td><td>Tyr</td><td>Arg</td><td>Glu</td><td>Arg</td><td>Leu</td><td>Gly</td><td>Leu</td><td>Pro</td>

<td></td><td> 2 90</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>Pro</td><td>Ala</td><td>Tyr</td><td>Arg</td><td>Gly</td><td>Val</td><td>Thr</td><td>Gly</td><td>Phe</td><td>Ala</td><td>Phe</td><td>Gly</td><td>Leu</td><td>Gly</td><td>Val</td><td>Glu</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

<td>Arg</td><td>Leu</td><td>Ala</td><td>Met</td><td>Leu</td><td>Arg</td><td>Tyr</td><td>Gly</td><td>Ile</td><td>Pro</td><td>Asp</td><td>Ile</td><td>Arg</td><td>Tyr</td><td>Phe</td><td>Phe</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td>

<td>Gly</td><td>Gly</td><td>Arg</td><td>Leu</td><td>Lys</td><td>Phe</td><td>Leu</td><td>Glu</td><td>Gin</td><td>Phe</td><td>Lys</td><td>Gly</td><td>Val</td><td>Leu</td><td></td><td></td>

340 345 350 &lt;210&gt; 70 &lt;211&gt; 350 &lt;212&gt; PRT &lt;213&gt; Thermus thermophilus &lt;400&gt; 70

<td>Mec 1</td><td>Leu</td><td>Glu</td><td>Glu</td><td>Ala 5</td><td>Leu</td><td>Ala</td><td>Ala</td><td>Ile</td><td>Gin 10</td><td>Asn</td><td>Ala</td><td>Arg</td><td>Asp</td><td>Leu 15</td><td>Glu</td>

<td>Glu</td><td>Leu</td><td>Lys</td><td>Ala 20</td><td>Leu</td><td>Lys</td><td>Ala</td><td>Arg</td><td>Tyr 25</td><td>Leu</td><td>Gly</td><td>Lys</td><td>Lys</td><td>Gly 30</td><td>Leu</td><td>Leu</td>

<td>Thr</td><td>Gin</td><td>Glu 35</td><td>Met</td><td>Lys</td><td>Gly</td><td>Leu</td><td>Ser 40</td><td>Ala</td><td>Leu</td><td>Pro</td><td>Leu</td><td>Glu 45</td><td>Glu</td><td>Arg</td><td>Arg</td>

<td>Lys</td><td>Arg 50</td><td>Gly</td><td>Gin</td><td>Glu</td><td>Leu</td><td>Asn 55</td><td>Ala</td><td>Ile</td><td>Lys</td><td>Ala</td><td>Ala 60</td><td>Leu</td><td>Glu</td><td>Ala</td><td>Ala</td>

<td>Leu 65</td><td>Glu</td><td>Ala</td><td>Arg</td><td>Glu</td><td>Lys 70</td><td>Ala</td><td>Leu</td><td>Glu</td><td>Glu</td><td>Ala 75</td><td>Ala</td><td>Leu</td><td>Lys</td><td>Glu</td><td>Ala 60</td>

<td>Leu</td><td>Glu</td><td>Arg</td><td>Glu</td><td>Arg 85</td><td>Val</td><td>Asp</td><td>Val</td><td>Ser</td><td>Leu 90</td><td>Pro</td><td>Gly</td><td>Ala</td><td>Ser</td><td>Leu 95</td><td>Phe</td>

<td>Ser</td><td>Gly</td><td>Gly</td><td>Leu 100</td><td>His</td><td>Pro</td><td>Ile</td><td>Thr</td><td>Leu 105</td><td>Met</td><td>Glu</td><td>Arg</td><td>Glu</td><td>Leu 110</td><td>val</td><td>Glu</td>

<td>Tle</td><td>Phe</td><td>Arg 115</td><td>Ala</td><td>Leu</td><td>Gly</td><td>Tyr</td><td>Gin 120</td><td>Ala</td><td>Val</td><td>Glu</td><td>Gly</td><td>Pro 125</td><td>Glu</td><td>Val</td><td>Glu</td>

<td>ser</td><td>Glu 130</td><td>Phe</td><td>Phe</td><td>Asn</td><td>Phe</td><td>Asp 135</td><td>Ala</td><td>Leu</td><td>Asn</td><td>Ile</td><td>Pro 140</td><td>Glu</td><td>His</td><td>His</td><td>Pro</td>

<td>Ala 145</td><td>Arg</td><td>Asp</td><td>Met</td><td>Trp</td><td>Asp 150</td><td>Thr</td><td>Phe</td><td>Trp</td><td>Leu</td><td>Thr 155</td><td>Gly</td><td>Glu</td><td>Gly</td><td>Phe</td><td>Arg 160</td>

<td>Leu</td><td>Glu</td><td>Gly</td><td>Pro</td><td>Leu 165</td><td>Gly</td><td>Glu</td><td>Glu</td><td>Val</td><td>Glu 170</td><td>Gly</td><td>Arg</td><td>Leu</td><td>Leu</td><td>Leu 175</td><td>Arg</td>

<td>Thr</td><td>His</td><td>Thr</td><td>Ser</td><td>Pro</td><td>Met</td><td>Gin</td><td>Val</td><td>Arg</td><td>Tyr</td><td>Met</td><td>Val</td><td>Ala</td><td>His</td><td>Thr</td><td>Pro</td>

-140-

<td>Pro</td><td>Phe</td><td>Arg</td><td>ISO Ile</td><td>Val</td><td>Val</td><td>Pro</td><td>Gly</td><td>1B5 Arg</td><td>Val</td><td>Phe</td><td>Arg</td><td>Phe</td><td>190 Glu</td><td>Gin</td><td>Thr</td>

<td>Aap</td><td>Ala</td><td>195 Thr</td><td>His</td><td>Glu</td><td>Ala</td><td>Val</td><td>200 Phe</td><td>His</td><td>Gin</td><td>Leu</td><td>Glu</td><td>205 Gly</td><td>Leu</td><td>Val</td><td>Val</td>

<td>Gly</td><td>210 Glu</td><td>Gly</td><td>Ile</td><td>Ala</td><td>Met</td><td>215 Ala</td><td>His</td><td>Leu</td><td>Lys</td><td>Gly</td><td>220 Ala</td><td>Ile</td><td>Tyr</td><td>Glu</td><td>Leu</td>

<td>225 Ala</td><td>Gin</td><td>Ala</td><td>Leu</td><td>Phe</td><td>230 Gly</td><td>Pro</td><td>Asp</td><td>Ser</td><td>Lys</td><td>235 Val</td><td>Arg</td><td>Phe</td><td>Gin</td><td>Pro</td><td>240 val</td>

<td>Tyr</td><td>Phe</td><td>Pro</td><td>Phe</td><td>245 Val</td><td>Glu</td><td>Pro</td><td>Gly</td><td>Ala</td><td>250 Gin</td><td>Phe</td><td>Ala</td><td>Val</td><td>Trp</td><td>255 Trp</td><td>Pro</td>

<td>Glu</td><td>Gly</td><td>Gly</td><td>260 Lys</td><td>Trp</td><td>Leu</td><td>Glu</td><td>Leu</td><td>265 Gly</td><td>Gly</td><td>Ala</td><td>Gly</td><td>Met</td><td>270 Val</td><td>His</td><td>Pro</td>

<td>Lys</td><td>Val</td><td>275 Phe</td><td>Gin</td><td>Ala</td><td>Val</td><td>Asp</td><td>280 Ala</td><td>Tyr</td><td>Arg</td><td>Glu</td><td>Arg</td><td>285 Leu</td><td>Gly</td><td>Leu</td><td>Pro</td>

<td>Pro</td><td>290 Ala</td><td>Tyr</td><td>Arg</td><td>Gly</td><td>Val</td><td>295 Thr</td><td>Gly</td><td>Phe</td><td>Ala</td><td>Phe</td><td>300 Gly</td><td>Leu</td><td>Gly</td><td>val</td><td>Glu</td>

<td>305 Arg</td><td>Leu</td><td>Ala</td><td>Met</td><td>Leu</td><td>310 Arg</td><td>Tyr</td><td>Gly</td><td>Ile</td><td>Pro</td><td>315 Asp</td><td>Ile</td><td>Arg</td><td>Tyr</td><td>Phe</td><td>320 Phe</td>

<td>Gly</td><td>Gly</td><td>Arg</td><td>Leu 340</td><td>325 Lys</td><td>Phe</td><td>Leu</td><td>Glu</td><td>Gin 345</td><td>330 Phe</td><td>Lys</td><td>Gly</td><td>Val</td><td>Leu 350</td><td> 335</td><td></td>

&lt;210&gt; 71 &lt;211&gt; 344 &lt;212&gt; PRT &lt;213&gt; Bacillus subtilis &lt;400&gt; 71

<td>Met 1</td><td>Glu</td><td>Glu</td><td>Lys</td><td>Leu 5</td><td>Lys</td><td>Gin</td><td>Leu</td><td>Glu</td><td>Gin 10</td><td>Glu</td><td>Ala</td><td>Leu</td><td>Glu</td><td>Gin 15</td><td>val</td>

<td>Glu</td><td>Ala</td><td>Ala</td><td>Ser 20</td><td>Ser</td><td>Leu</td><td>Lys</td><td>Val</td><td>Val 25</td><td>Asn</td><td>Asp</td><td>Ile</td><td>Arg</td><td>Val 30</td><td>Gin</td><td>Tyr</td>

<td>Leu</td><td>Gly</td><td>Lys 35</td><td>Lys</td><td>Gly</td><td>Pro</td><td>Ile</td><td>Thr 40</td><td>Glu</td><td>Val</td><td>Leu</td><td>Arg</td><td>Gly 45</td><td>Met</td><td>Gly</td><td>Lys</td>

<td>Leu</td><td>ser 50</td><td>Ala</td><td>Glu</td><td>Glu</td><td>Arg</td><td>pro 55</td><td>Lys</td><td>Met</td><td>Gly</td><td>Ala</td><td>Leu 60</td><td>Ala</td><td>Asn</td><td>Glu</td><td>Val</td>

<td>Arg 65</td><td>Glu</td><td>Arg</td><td>Ile</td><td>Ala</td><td>Asn 70</td><td>Ala</td><td>Ile</td><td>Ala</td><td>Asp</td><td>Lys 75</td><td>Asn</td><td>Glu</td><td>Lys</td><td>Leu</td><td>Glu 80</td>

<td>Glu</td><td>Glu</td><td>Glu</td><td>Met</td><td>Lys 85</td><td>Gin</td><td>Lys</td><td>Leu</td><td>Ala</td><td>Gly 90</td><td>Gin</td><td>Thr</td><td>Ile</td><td>Asp</td><td>val 95</td><td>Thr</td>

<td>Leu</td><td>Pro</td><td>Gly</td><td>Asn 100</td><td>Pro</td><td>Val</td><td>Ala</td><td>Val</td><td>Gly 105</td><td>Gly</td><td>Arg</td><td>His</td><td>Pro</td><td>Leu 110</td><td>Thr</td><td>Val</td>

<td>val</td><td>Ile</td><td>Glu 115</td><td>Glu</td><td>Ile</td><td>Glu</td><td>Asp</td><td>Leu 120</td><td>Phe</td><td>Ile</td><td>Gly</td><td>Met</td><td>Gly 12S</td><td>Tyr</td><td>Thr</td><td>Val</td>

<td>Glu</td><td>Glu 130</td><td>Gly</td><td>Pro</td><td>Glu</td><td>val</td><td>Glu 135</td><td>Thr</td><td>Asp</td><td>Tyr</td><td>Tyr</td><td>Asn 140</td><td>Phe</td><td>Glu</td><td>Ser</td><td>Leu</td>

<td>Asn 145</td><td>Leu</td><td>Pro</td><td>Lys</td><td>Glu</td><td>His 150</td><td>Pro</td><td>Ala</td><td>Arg</td><td>Asp</td><td>Met 155</td><td>Gin</td><td>Asp</td><td>Ser</td><td>Phe</td><td>Tyr 160</td>

<td>ile</td><td>Thr</td><td>Glu</td><td>Glu</td><td>Thr 16S</td><td>Leu</td><td>Met</td><td>Arg</td><td>Thr</td><td>Gin 170</td><td>Thr</td><td>Ser</td><td>Pro</td><td>Val</td><td>Gin 175</td><td>Thr</td>

<td>Arg</td><td>Thr</td><td>Met</td><td>Glu ISO</td><td>Lys</td><td>His</td><td>Glu</td><td>Gly</td><td>Lys 185</td><td>Gly</td><td>Pro</td><td>Val</td><td>Lys</td><td>Ile 190</td><td>Ile</td><td>Cys</td>

<td>Pro</td><td>Gly</td><td>Lys 195</td><td>Val</td><td>Tyr</td><td>Arg</td><td>Arg</td><td>Asp 200</td><td>Asn</td><td>Asp</td><td>Asp</td><td>Ala</td><td>Thr 205</td><td>His</td><td>Ser</td><td>His</td>

<td>Gin</td><td>Phe 210</td><td>Met</td><td>Gin</td><td>Ile</td><td>Glu</td><td>Gly 215</td><td>Leu</td><td>Val</td><td>Val</td><td>Asp</td><td>Lys 220</td><td>Asn</td><td>Ile</td><td>Ser</td><td>Met</td>

<td>Ser</td><td>Asp</td><td>Leu</td><td>Lys</td><td>Gly</td><td>Thr</td><td>Leu</td><td>Glu</td><td>Leu</td><td>Val</td><td>Ala</td><td>Lys</td><td>Lys</td><td>Met</td><td>Phe</td><td>Gly</td>

-141-

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>Gln</td><td>Asp</td><td>Arg</td><td>Glu</td><td>Ile</td><td>Arg</td><td>Leu</td><td>Alt,*</td><td>Pro</td><td>Ser</td><td>Phe</td><td>Phe</td><td>Pro</td><td>Phe</td><td>Thr</td><td>Glu</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

<td>pro</td><td>Ser</td><td>Val</td><td>Glu</td><td>val</td><td>Asp</td><td>val</td><td>Thr</td><td>Cys</td><td>Phe</td><td>Lys</td><td>Cys</td><td colspan="2">Gly Gly</td><td>Asn</td><td>Gly</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>Cys</td><td>Ser</td><td>Val</td><td>Cys</td><td>Lys</td><td>Gly</td><td>Thr</td><td>Gly</td><td>Trp</td><td>Ile</td><td>Glu</td><td>Ile</td><td>Leu</td><td>Gly</td><td>Ala</td><td>Gly</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>Met</td><td>Val</td><td>His</td><td>Pro</td><td>Asn</td><td>Val</td><td>Leu</td><td>Lys</td><td>Met</td><td>Ala</td><td>Gly</td><td>Phe</td><td>Asp</td><td>Pro</td><td>Lys</td><td>Glu</td>

<td></td><td> 2 90</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>Tyr</td><td>Gln</td><td>Gly</td><td>Phe</td><td>Ala</td><td>Phe</td><td>Gly</td><td>Met</td><td>Gly</td><td>val</td><td>Glu</td><td>Arg</td><td>Ile</td><td>Ala</td><td>Met</td><td>Leu</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

<td>Lys</td><td>Tyr</td><td>Gly</td><td>Ile</td><td>Asp</td><td>Asp</td><td>Ile</td><td>Arg</td><td>His</td><td>Phe</td><td>Tyr</td><td>Thr</td><td>Asn</td><td>Asp</td><td>Val</td><td>Arg</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td>

<td>Phe</td><td>Ile</td><td>Ser</td><td>Gln</td><td>Phe</td><td>Lys</td><td>Gln</td><td>Ala</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

340 &lt;210&gt; 72 &lt;211&gt; 328 &lt;212&gt; PRT &lt;213&gt; Helicobacter pylon &lt;400&gt; 72

<td rowspan="2">Met 1</td><td colspan="10">His Thr Leu Ile Glu Arg Leu Glu Lys Val</td><td colspan="2" rowspan="2">Thr Asn</td><td rowspan="2">Ser</td><td rowspan="2">Lys 15</td><td rowspan="2">Glu</td>

<td colspan="7"> 5</td><td colspan="3"> 10</td>

<td>Leu</td><td>Glu</td><td>Glu</td><td>Ala</td><td>Arg</td><td>Leu</td><td>Asn</td><td>Ala</td><td>Leu</td><td>Gly</td><td>Lys</td><td>Lys</td><td>Gly</td><td>Val</td><td>Phe</td><td>Ala</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Asp</td><td>Lys</td><td>Phe</td><td>Asn</td><td>Gin</td><td>Leu</td><td>Lys</td><td>His</td><td>Leu</td><td>Asn</td><td>Gly</td><td>Glu</td><td>Glu</td><td>Lys</td><td>Asn</td><td>Ala</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Phe</td><td>Ala</td><td>Lys</td><td>Glu</td><td>Ile</td><td>His</td><td>His</td><td>Tyr</td><td>Lys</td><td>Gln</td><td>Ala</td><td>Phe</td><td>Glu</td><td>Lys</td><td>Ala</td><td>Phe</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Glu</td><td>Trp</td><td>Lys</td><td>Lys</td><td>Lys</td><td>Ala</td><td>Ile</td><td>Ile</td><td>Glu</td><td>Leu</td><td>Glu</td><td>Leu</td><td>Glu</td><td>Glu</td><td>Arg</td><td>Leu</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Lys</td><td>Lys</td><td>Glu</td><td>Lys</td><td>Ile</td><td>Asp</td><td>Val</td><td>Ser</td><td>Leu</td><td>Phe</td><td>Asn</td><td>Ala</td><td>Ile</td><td>Lys</td><td>Thr</td><td>Ser</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Ser</td><td>Ser</td><td>His</td><td>Pro</td><td>Leu</td><td>Αβη</td><td>Tyr</td><td>Thr</td><td>Lys</td><td>Asn</td><td>Lys</td><td>ile</td><td>Ile</td><td>Glu</td><td>Phe</td><td>Phe</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Thr</td><td>Pro</td><td>Leu</td><td>Gly</td><td>Tyr</td><td>Lys</td><td>Leu</td><td>Glu</td><td>Ile</td><td>Gly</td><td>Ser</td><td>Leu</td><td>Val</td><td>Glu</td><td>Asp</td><td>Asp</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Phe</td><td>His</td><td>Asn</td><td>Phe</td><td>Ser</td><td>Ala</td><td>Leu</td><td>Asn</td><td>Leu</td><td>Pro</td><td>Pro</td><td>Tyr</td><td>His</td><td>Pro</td><td>Ala</td><td>Arg</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Asp</td><td>Met</td><td>Gln</td><td>Asp</td><td>Thr</td><td>Phe</td><td>Tyr</td><td>Phe</td><td>Lys</td><td>Asp</td><td>His</td><td>Lys</td><td>Leu</td><td>Leu</td><td>Arg</td><td>Thr</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>His</td><td>Thr</td><td>Ser</td><td>Pro</td><td>val</td><td>Gln</td><td>Ile</td><td>His</td><td>Thr</td><td>Met</td><td>Gln</td><td>Glu</td><td>Gln</td><td>Thr</td><td>Pro</td><td>Pro</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Ile</td><td>Lys</td><td>Met</td><td>Ile</td><td>Cys</td><td>Leu</td><td>Gly</td><td>Glu</td><td>Thr</td><td>Phe</td><td>Arg</td><td>Arg</td><td>Asp</td><td>Tyr</td><td>Asp</td><td>Leu</td>

<td></td><td></td><td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Thr</td><td>His</td><td>Thr</td><td>Pro</td><td>Met</td><td>Phe</td><td>His</td><td>Gln</td><td>Ile</td><td>Glu</td><td>Gly</td><td>Leu</td><td>Val</td><td>Val</td><td>Asp</td><td>Gln</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Ly9</td><td>Gly</td><td>Asn</td><td>Ile</td><td>Arg</td><td>Phe</td><td>Thr</td><td>His</td><td>Leu</td><td>Lys</td><td>Gly</td><td>val</td><td>Ile</td><td>Glu</td><td>ASp</td><td>Phe</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Leu</td><td>His</td><td>Tyr</td><td>Phe</td><td>Phe</td><td>Gly</td><td>Gly</td><td>Val</td><td>Lys</td><td>Leu</td><td>Arg</td><td>Trp</td><td>Arg</td><td>Ser</td><td>ser</td><td>Phe</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>Phe</td><td>Pro</td><td>Phe</td><td>Thr</td><td>Glu</td><td>Pro</td><td>Ser</td><td>Ala</td><td>Glu</td><td>Val</td><td>Asp</td><td>Ile</td><td>Ser</td><td>Cys</td><td>Val</td><td>Phe</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

<td>cys</td><td>Lys</td><td>Gln</td><td>Glu</td><td>Gly</td><td>Cys</td><td>Arg</td><td>Val</td><td>Cys</td><td>Ser</td><td>His</td><td>Thr</td><td>Gly</td><td>Trp</td><td>Leu</td><td>Glu</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>Val</td><td>Leu</td><td>Gly</td><td>Cys</td><td>Gly</td><td>Met</td><td>Val</td><td>Asn</td><td>Asn</td><td>Ala</td><td>val</td><td>Phe</td><td>Glu</td><td>Ala</td><td>Ile</td><td>Gly</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>Tyr</td><td>Glu</td><td>Asn</td><td>Val</td><td>Ser</td><td>Gly</td><td>Phe</td><td>Ala</td><td>Phe</td><td>Gly</td><td>Met</td><td>Gly</td><td>Ile</td><td>Glu</td><td>Arg</td><td>Leu</td>

<td></td><td> 2 90</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>Ala</td><td>Met</td><td>Leu</td><td>Thr</td><td>Cys</td><td>Gln</td><td>ile</td><td>Asn</td><td>Asp</td><td>Leu</td><td>Arg</td><td>Ser</td><td>Phe</td><td>phe</td><td>Glu</td><td>Thr</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

<td>Asp</td><td>Leu</td><td>Arg</td><td>Val</td><td>Leu</td><td>Glu</td><td>Ser</td><td>Phe</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 73 &lt;211&gt; 327

-142&lt;212&gt; PRT &lt;213&gt; Escherichia coli &lt;400&gt; 73

<td>Met 1</td><td>Ser</td><td>His</td><td>Leu</td><td>Ala 5</td><td>Glu</td><td>Leu</td><td>Val</td><td>Ala</td><td>Ser 10</td><td>Ala</td><td>Lys</td><td>Ala</td><td>Ala</td><td>Ile 15</td><td>Ser</td>

<td>Gin</td><td>Ala</td><td>Ser</td><td>Asp 20</td><td>Val</td><td>Ala</td><td>Ala</td><td>Leu</td><td>Asp 25</td><td>Asn</td><td>Val</td><td>Arg</td><td>Val</td><td>Glu 30</td><td>Tyr</td><td>Leu</td>

<td>Gly</td><td>Lys</td><td>Lys 35</td><td>Gly</td><td>His</td><td>Leu</td><td>Thr</td><td>Leu 40</td><td>Gin</td><td>Met</td><td>Thr</td><td>Thr</td><td>Leu 45</td><td>Arg</td><td>Glu</td><td>Leu</td>

<td>Pro</td><td>Pro 50</td><td>Glu</td><td>Glu</td><td>Arg</td><td>Pro</td><td>Ala 55</td><td>Ala</td><td>Gly</td><td>Ala</td><td>Val</td><td>Ile 60</td><td>Asn</td><td>Glu</td><td>Ala</td><td>Lys</td>

<td>Glu 65</td><td>Gin</td><td>Val</td><td>Gin</td><td>Gin</td><td>Ala 70</td><td>Leu</td><td>Asn</td><td>Ala</td><td>Arg</td><td>Lys 75</td><td>Ala</td><td>Glu</td><td>Leu</td><td>Glu</td><td>Ser 80</td>

<td>Ala</td><td>Ala</td><td>Leu</td><td>Asn</td><td>Ala 85</td><td>Arg</td><td>Leu</td><td>Ala</td><td>Ala</td><td>Glu 90</td><td>Thr</td><td>Ile</td><td>Asp</td><td>Val</td><td>Ser 95</td><td>Leu</td>

<td>Pro</td><td>Gly</td><td>Arg</td><td>Arg 100</td><td>Ile</td><td>Glu</td><td>Asn</td><td>Gly</td><td>Gly 105</td><td>Leu</td><td>His</td><td>Pro</td><td>val</td><td>Thr 110</td><td>Arg</td><td>Thr</td>

<td>Ile</td><td>Asp</td><td>Arg 115</td><td>Ile</td><td>Glu</td><td>Ser</td><td>Phe</td><td>Phe 120</td><td>Gly</td><td>Glu</td><td>Leu</td><td>Gly</td><td>Phe 125</td><td>Thr</td><td>Val</td><td>Ala</td>

<td>Thr</td><td>Gly 130</td><td>Pro</td><td>Glu</td><td>ile</td><td>Glu</td><td>Asp 135</td><td>Asp</td><td>Tyr</td><td>His</td><td>Asn</td><td>Phe 140</td><td>Asp</td><td>Ala</td><td>Leu</td><td>Asn</td>

<td>Ile 145</td><td>Pro</td><td>Gly</td><td>Hi3</td><td>His</td><td>Pro 150</td><td>Ala</td><td>Arg</td><td>Ala</td><td>Asp</td><td>Hi 3 155</td><td>Asp</td><td>Thr</td><td>Phe</td><td>Trp</td><td>Phe 160</td>

<td>Asp</td><td>Thr</td><td>Thr</td><td>Arg</td><td>Leu 165</td><td>Leu</td><td>Arg</td><td>Thr</td><td>Gin</td><td>Thr 170</td><td>ser</td><td>Gly</td><td>val</td><td>Gin</td><td>Ile 175</td><td>Arg</td>

<td>Thr</td><td>Met</td><td>Lys</td><td>Ala ISO</td><td>Gin</td><td>Gin</td><td>Pro</td><td>Pro</td><td>Ile 185</td><td>Arg</td><td>Ile</td><td>Ile</td><td>Ala</td><td>Pro 190</td><td>Gly</td><td>Arg</td>

<td>Val</td><td>Tyr</td><td>Arg 19S</td><td>Asn</td><td>Asp</td><td>Tyr</td><td>Asp</td><td>Gin 200</td><td>Thr</td><td>His</td><td>Thr</td><td>Pro</td><td>Met 20S</td><td>Phe</td><td>His</td><td>Gin</td>

<td>Met</td><td>Glu 210</td><td>Gly</td><td>Leu</td><td>Ile</td><td>Val</td><td>Asp 215</td><td>Thr</td><td>Asn</td><td>Ile</td><td>Ser</td><td>Phe 220</td><td>Thr</td><td>Asn</td><td>Leu</td><td>Lys</td>

<td>Gly 225</td><td>Thr</td><td>Leu</td><td>His</td><td>Asp</td><td>Phe 230</td><td>Leu</td><td>Arg</td><td>Asn</td><td>Phe</td><td>Phe 235</td><td>Glu</td><td>Glu</td><td>Asp</td><td>Leu</td><td>Gin 240</td>

<td>Ile</td><td>Arg</td><td>Phe</td><td>Arg</td><td>Pro 245</td><td>Ser</td><td>Tyr</td><td>Phe</td><td>Pro</td><td>Phe 250</td><td>Thr</td><td>Glu</td><td>Pro</td><td>Ser</td><td>Ala 255</td><td>Glu</td>

<td>Val</td><td>Asp</td><td>Val</td><td>Met 260</td><td>Gly</td><td>Lys</td><td>Asn</td><td>Gly</td><td>Lys 265</td><td>Trp</td><td>Leu</td><td>Glu</td><td>val</td><td>Leu 270</td><td>Gly</td><td>Cys</td>

<td>Gly</td><td>Met</td><td>Val 275</td><td>His</td><td>Pro</td><td>Asn</td><td>val</td><td>Leu 280</td><td>Arg</td><td>Asn</td><td>val</td><td>Gly</td><td>Ile 285</td><td>Asp</td><td>Pro</td><td>Glu</td>

<td>Val</td><td>Tyr 290</td><td>Ser</td><td>Gly</td><td>Phe</td><td>Ala</td><td>Phe 295</td><td>Gly</td><td>Met</td><td>Gly</td><td>Met</td><td>Glu 300</td><td>Arg</td><td>Leu</td><td>Thr</td><td>Met</td>

<td>Leu 305 Arg</td><td>Arg Phe</td><td>Tyr Leu</td><td>Gly Lys</td><td>Val Gin</td><td>Thr 310 Phe</td><td>Asp Lys</td><td>Leu</td><td>Arg</td><td>Ser</td><td>Phe 315</td><td>Phe</td><td>Glu</td><td>Asn</td><td>ASp</td><td>Leu 320</td>

32S &lt;210&gt; 74 &lt;211&gt; 453 &lt;212&gt; PRT &lt;213&gt; Drosophila melanogaster &lt;400&gt; 74

-143-

<td>Met</td><td>Leu</td><td>Leu</td><td>Thr</td><td>Leu</td><td>Arg</td><td>val</td><td>Gin</td><td>Gly</td><td>Ala</td><td>Arg</td><td>His</td><td>Trp</td><td>Leu</td><td>Lys</td><td>Ser</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Thr</td><td>Arg</td><td>Cys</td><td>Leu</td><td>Ala</td><td>Ser</td><td>Ser</td><td>Ala</td><td>Ala</td><td>Pro</td><td>Ala</td><td>Lys</td><td>ser</td><td>Pro</td><td>Ser</td><td>Ser</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Pro</td><td>Pro</td><td>Gin</td><td>Leu</td><td>Glu</td><td>Val</td><td>Ser</td><td>Gly</td><td>Ser</td><td>Thr</td><td>Tyr</td><td>Ala</td><td>Thr</td><td>Asp</td><td>Gly</td><td>Trp</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Thr</td><td>Asn</td><td>Val</td><td>Thr</td><td>Pro</td><td>Lys</td><td>Ile</td><td>Leu</td><td>Ser</td><td>Tyr</td><td>Val</td><td>Gly</td><td>Ala</td><td>Asn</td><td>Lys</td><td>His</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Leu</td><td>Gin</td><td>Thr</td><td>Asp</td><td>Kis</td><td>Pro</td><td>Leu</td><td>Ser</td><td>Ile</td><td>Ile</td><td>Arg</td><td>Gin</td><td>Arg</td><td>Ile</td><td>Val</td><td>Asn</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Tyr</td><td>Phe</td><td>Tyr</td><td>Gly</td><td>Ala</td><td>Tyr</td><td>Arg</td><td>Asn</td><td>Gin</td><td>Arg</td><td>Gly</td><td>Asn</td><td>Pro</td><td>Leu</td><td>Phe</td><td>Ser</td>

90 95

Val Tyr Asp Gin Met Asn Pro Val Val Thr Val Gin Gin Asn Phe Asp 100 105 110

<td colspan="2">Asn Leu</td><td>Leu 115</td><td>Ile</td><td>Pro</td><td>Ala</td><td colspan="3">Asp His Val 120</td><td>Ser</td><td>Arg</td><td>Gin</td><td colspan="4">Lys Ser Asp Cys 125</td>

<td>Tyr</td><td>Tyr</td><td>Ile</td><td>Asn</td><td>Gin</td><td>Gin</td><td>His</td><td>Leu</td><td>Leu</td><td>Arg</td><td>Ala</td><td>His</td><td>Thr</td><td>Thr</td><td>Ala</td><td>His</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 14 0</td><td></td><td></td><td></td><td></td>

<td>Gin</td><td>Val</td><td>Glu</td><td>Leu</td><td>ile</td><td>Ser</td><td>Gly</td><td>Gly</td><td>Leu</td><td>Asp</td><td>Asn</td><td>Phe</td><td>Leu</td><td>val</td><td>val</td><td>Gly</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Glu</td><td>Val</td><td>Tyr</td><td>Arg</td><td>Arg</td><td>Asp</td><td>GlU</td><td>Ile</td><td>Asp</td><td>Ser</td><td>Thr</td><td>His</td><td>Tyr</td><td>Pro</td><td>Val</td><td>Phe</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>His</td><td>Gin</td><td>Ala</td><td>Asp</td><td>Ala</td><td>Val</td><td>Arg</td><td>Leu</td><td>Val</td><td>Thr</td><td>Lys</td><td>Asp</td><td>Lys</td><td>Leu</td><td>Phe</td><td>Glu</td>

<td></td><td></td><td></td><td>ISO</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Arg</td><td>Asn</td><td>Pro</td><td>Gly</td><td>Leu</td><td>Glu</td><td>Leu.</td><td>Phe</td><td>Glu</td><td>Glu</td><td>Thr</td><td>Trp</td><td>Ser</td><td>Gly</td><td>Thr</td><td>Leu</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Ala</td><td>Asp</td><td>Pro</td><td>Lys</td><td>Leu</td><td>Ile</td><td>Leu</td><td>Pro</td><td>Ser</td><td>Ser</td><td>Lys</td><td>Phe</td><td>Met</td><td>Asp</td><td>Gin</td><td>Thr</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Lys</td><td>Gin</td><td>pro</td><td>Cys</td><td>His</td><td>Thr</td><td>Leu</td><td>Glu</td><td>Ala</td><td>Val</td><td>Lys</td><td>Leu</td><td>Met</td><td>Glu</td><td>His</td><td>Glu</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>Met</td><td>Lys</td><td>His</td><td>val</td><td>Leu</td><td>Val</td><td>Gly</td><td>Leu</td><td>Thr</td><td>Lys</td><td>Asp</td><td>Leu</td><td>Phe</td><td>Gly</td><td>Pro</td><td>Arg</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

<td>Ile</td><td>Lys</td><td>Tyr</td><td>Arg</td><td>Trp</td><td>Val</td><td>Asp</td><td>Thr</td><td>Tyr</td><td>Phe</td><td>Pro</td><td>Phe</td><td>Thr</td><td>Gin</td><td>Pro</td><td>Ser</td>

260 265 270

Trp Glu Leu Glu Ile Tyr Phe Lys Asp Asn Trp Leu Glu Val Leu Gly 275 280 285

Cys Gly Ile Met Arg His Glu Ile Leu Gin Arg Ser Gly Val His Gin 290 295 300

Ser Ile Gly Tyr Ala Phe Gly Val Gly Leu Glu Arg Leu Ala Met Val

<td>30S Leu</td><td>Phe</td><td>Asp</td><td>Ile</td><td>Pro</td><td>310 Asp</td><td>Ile</td><td>Arg</td><td>Leu</td><td>Phe</td><td>215 Trp</td><td>Ser</td><td>Asn</td><td>Asp</td><td>Ser</td><td>320 Gly</td>

<td>Phe</td><td>Leu</td><td>Ser</td><td>Gin</td><td>325 Phe</td><td>Ser</td><td>Glu</td><td>Lys</td><td>Asp</td><td>330 Leu</td><td>His</td><td>Asn</td><td>Leu</td><td>Pro</td><td>335 Lys</td><td>Tyr</td>

<td>Lys</td><td>Pro</td><td>Ile</td><td>340 Ser</td><td>His</td><td>Tyr</td><td>Pro</td><td>Gin</td><td>345 Cys</td><td>Thr</td><td>Asn</td><td>Asp</td><td>Leu</td><td>350 Ser</td><td>Phe</td><td>Trp</td>

<td>Leu</td><td>Pro</td><td>355 Gin</td><td>Asp</td><td>Ile</td><td>Glu</td><td>Val</td><td>360 Asp</td><td>Ala</td><td>Gly</td><td>Phe</td><td>Ser</td><td>365 Pro</td><td>Asn</td><td>Asp</td><td>Phe</td>

<td>Tyr</td><td>370 Asp</td><td>Lau</td><td>Val</td><td>Arg</td><td>Ser</td><td>375 Val</td><td>Ala</td><td>Gly</td><td>Asp</td><td>Met</td><td>380 Val</td><td>Glu</td><td>Gin</td><td>Ile</td><td>Ser</td>

<td>385 Leu</td><td>Val</td><td>Asp</td><td>Lys</td><td>Phe</td><td>390 Lys</td><td>His</td><td>PrO</td><td>Lys</td><td>Thr</td><td>395 Gly</td><td>L/3</td><td>ser</td><td>Ser</td><td>Val</td><td>400 Cys</td>

<td>Phe</td><td>Arg</td><td>Ile</td><td>Val</td><td>405 Tyr</td><td>Arg</td><td>His</td><td>Met</td><td>Glu</td><td>410 Arg</td><td>Thr</td><td>Leu</td><td>Thr</td><td>Gin</td><td>415 Ala</td><td>Glu</td>

<td>Val</td><td>Asn</td><td>Glu</td><td>420 Ile</td><td>His</td><td>Lys</td><td>Gin</td><td>Ile</td><td>425 Ala</td><td>Ser</td><td>Ala</td><td>Ser</td><td>Val</td><td>430 Asp</td><td>ser</td><td>Phe</td>

<td>Asn</td><td>Val 450</td><td>435 Gin</td><td>Ile</td><td>Arg</td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td></td><td></td><td></td>

&lt;210&gt; 75 &lt;211&gt; 339 &lt;212&gt; PRT &lt;213&gt; Chlamydophila pneumoniae &lt;400&gt; 75

-144-

<td>Met 1</td><td>Glu</td><td>Met</td><td>Lys</td><td>Glu 5</td><td>Glu</td><td>Ile</td><td>Glu</td><td>Ala</td><td>Val 10</td><td>Lys</td><td>Gin</td><td>Gin</td><td>Phe</td><td>His 15</td><td>Ser</td>

<td>Glu</td><td>Leu</td><td>Asp</td><td>Gin 20</td><td>Val</td><td>Asn</td><td>Ser</td><td>Ser</td><td>Gin 25</td><td>Ala</td><td>Leu</td><td>Ala</td><td>Asp</td><td>Leu 30</td><td>Lys</td><td>val</td>

<td>Arg</td><td>Tyr</td><td>Leu 35</td><td>Gly</td><td>Lys</td><td>Lys</td><td>Gly</td><td>Ile 40</td><td>Phe</td><td>Arg</td><td>Ser</td><td>phe</td><td>Ser 45</td><td>Glu</td><td>Lys</td><td>Leu</td>

<td>Lys</td><td>Gin 50</td><td>Cys</td><td>Thr</td><td>Asp</td><td>Lys</td><td>Ala 55</td><td>Lys</td><td>Leu</td><td>Gly</td><td>Ser</td><td>Leu 60</td><td>Ile</td><td>Asn</td><td>Asp</td><td>Phe</td>

<td>Lys 65</td><td>Thr</td><td>Tyr</td><td>Val</td><td>Glu</td><td>Asp 70</td><td>Leu</td><td>Leu</td><td>Gin</td><td>Glu</td><td>Lys 75</td><td>Ser</td><td>Leu</td><td>Val</td><td>Leu</td><td>Leu 80</td>

<td>Ala</td><td>Ser</td><td>Glu</td><td>Gin</td><td>Ala 85</td><td>Glu</td><td>Ala</td><td>phe</td><td>Ser</td><td>Lys 90</td><td>Glu</td><td>Lys</td><td>Ile</td><td>Asp</td><td>Ser 95</td><td>Ser</td>

<td>Leu</td><td>pro</td><td>Gly</td><td>Asp 100</td><td>ser</td><td>Gin</td><td>Pro</td><td>Ser</td><td>Gly 105</td><td>Gly</td><td>Arg</td><td>His</td><td>ile</td><td>Leu 110</td><td>Lys</td><td>Ser</td>

<td>Ile</td><td>Leu</td><td>Asp 115</td><td>Asp</td><td>val</td><td>Val</td><td>Asp</td><td>Ile 120</td><td>Phe</td><td>Val</td><td>His</td><td>Leu</td><td>Gly 125</td><td>Phe</td><td>Cys</td><td>Val</td>

<td>Arg</td><td>Glu 130</td><td>Ala</td><td>Pro</td><td>Aan</td><td>Ile</td><td>Glu 135</td><td>Ser</td><td>Glu</td><td>Ala</td><td>Asn</td><td>Asn 14 0</td><td>Phe</td><td>Thr</td><td>Leu</td><td>Leu</td>

<td>Asn 145</td><td>Phe</td><td>Thr</td><td>Glu</td><td>Asp</td><td>His 150</td><td>Pro</td><td>Ala</td><td>Arg</td><td>Gin</td><td>Met 155</td><td>Kis</td><td>Asp</td><td>Thr</td><td>Phe</td><td>Tyr 160</td>

<td>Leu</td><td>Aan</td><td>Ala</td><td>Thr</td><td>Thr 165</td><td>Val</td><td>Leu</td><td>Arg</td><td>Thr</td><td>His 170</td><td>Thr</td><td>Ser</td><td>Asn</td><td>Val</td><td>Gin 175</td><td>Ala</td>

<td>Arg</td><td>Glu</td><td>Leu</td><td>Lys ISO</td><td>Lys</td><td>Gin</td><td>Gin</td><td>Pro</td><td>Pro 185</td><td>Ile</td><td>Lys</td><td>val</td><td>Val</td><td>Ala 190</td><td>Pro</td><td>Gly</td>

<td>Leu</td><td>Cys</td><td>Phe 195</td><td>Arg</td><td>Asn</td><td>Glu</td><td>Asp</td><td>Ile 200</td><td>Ser</td><td>Ala</td><td>Arg</td><td>ser</td><td>His 205</td><td>Val</td><td>Leu</td><td>Phe</td>

<td>His</td><td>Gin 210</td><td>Val</td><td>Glu</td><td>Ala</td><td>Phe</td><td>Tyr 215</td><td>Val</td><td>Asp</td><td>His</td><td>Asn</td><td>val 220</td><td>Thr</td><td>Phe</td><td>Ser</td><td>Asp</td>

<td>Leu 225</td><td>Thr</td><td>Ala</td><td>Ile</td><td>Leu</td><td>Ser 230</td><td>Ala</td><td>Phe</td><td>Tyr</td><td>His</td><td>ser 235</td><td>Phe</td><td>Phe</td><td>Gin</td><td>Arg</td><td>Lys 24 0</td>

<td>Thr</td><td>Glu</td><td>Leu</td><td>Arg</td><td>Phe 245</td><td>Arg</td><td>His</td><td>Ser</td><td>Tyr</td><td>Phe 250</td><td>Pro</td><td>Phe</td><td>Val</td><td>Glu</td><td>Pro 255</td><td>Gly</td>

<td>Ile</td><td>Glu</td><td>Val</td><td>Asp 260</td><td>Val</td><td>Ser</td><td>Cys</td><td>Glu</td><td>Cys 265</td><td>Cys</td><td>Gly</td><td>Lys</td><td>Gly</td><td>Cys 270</td><td>Ala</td><td>Leu</td>

<td>Cys</td><td>Lys</td><td>His 275</td><td>Thr</td><td>Gly</td><td>Trp</td><td>Leu</td><td>Glu 280</td><td>val</td><td>Ala</td><td>Gly</td><td>Ala</td><td>Gly 285</td><td>Met</td><td>Ile</td><td>His</td>

<td>Pro</td><td>Gin 2 90</td><td>Val</td><td>Leu</td><td>Arg</td><td>Asn</td><td>Gly 295</td><td>Asn</td><td>Val</td><td>Asp</td><td>Pro</td><td>Glu 300</td><td>Ile</td><td>Tyr</td><td>ser</td><td>Gly</td>

<td>Tyr 305</td><td>Ala</td><td>val</td><td>Gly</td><td>Met</td><td>Gly 310</td><td>Ile</td><td>Glu</td><td>Arg</td><td>Leu</td><td>Ala 315</td><td>Met</td><td>Leu</td><td>Lys</td><td>Tyr</td><td>Gly 320</td>

<td>Val Gin</td><td>Ser Phe</td><td>Asp Ser</td><td>Ile</td><td>Arg 325</td><td>Leu</td><td>Phe</td><td>Ser</td><td>Glu</td><td>Asn 330</td><td>Asp</td><td>Leu</td><td>Arg</td><td>Phe</td><td>Leu 335</td><td>Gin</td>

&lt;210&gt; 76 &lt;211&gt; 451 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 76

-145-

<td>Met 1</td><td>Val</td><td>Gly</td><td>Ser</td><td>Ala 5</td><td>Leu</td><td>Arg</td><td>Arg</td><td>Gly</td><td>Ala 10</td><td>His</td><td>Ala</td><td>Tyr</td><td>Val</td><td>Tyr 15</td><td>Leu</td>

<td>Val</td><td>Ser</td><td>Lys</td><td>Ala 20</td><td>Ser</td><td>His</td><td>Ile</td><td>Ser</td><td>Arg 25</td><td>Gly</td><td>His</td><td>Gin</td><td>His</td><td>Gin 30</td><td>Ala</td><td>Trp</td>

<td>Gly</td><td>ser</td><td>Arg 35</td><td>Pro</td><td>Pro</td><td>Ala</td><td>Ala</td><td>Glu 40</td><td>cys</td><td>Ala</td><td>Thr</td><td>Gin</td><td>Arg 45</td><td>Ala</td><td>Pro</td><td>Gly</td>

<td>Ser</td><td>Val 50</td><td>Val</td><td>Glu</td><td>Leu</td><td>Leu</td><td>Gly 55</td><td>Lys</td><td>Ser</td><td>Tyr</td><td>Pro</td><td>Gin 60</td><td>Asp</td><td>Asp</td><td>His</td><td>Ser</td>

<td>Asn 65</td><td>Leu</td><td>Thr</td><td>Arg</td><td>Lys</td><td>Val 70</td><td>Leu</td><td>Thr</td><td>Arg</td><td>Val</td><td>Gly 75</td><td>Arg</td><td>Asn</td><td>Leu</td><td>His</td><td>Asn 80</td>

<td>Gin</td><td>Gin</td><td>His</td><td>His</td><td>Pro 85</td><td>Leu</td><td>Trp</td><td>Leu</td><td>Ile</td><td>Lys 90</td><td>Glu</td><td>Arg</td><td>Val</td><td>Lys</td><td>Glu 95</td><td>His</td>

<td>Phe</td><td>Tyr</td><td>Lys</td><td>Gin 100</td><td>Tyr</td><td>Val</td><td>Gly</td><td>Arg</td><td>Phe 105</td><td>Gly</td><td>Thr</td><td>Pro</td><td>Leu</td><td>Phe 110</td><td>Ser</td><td>Val</td>

<td>Tyr</td><td>Asp</td><td>Asn 115</td><td>Leu</td><td>Ser</td><td>Pro</td><td>Val</td><td>val 120</td><td>Thr</td><td>Thr</td><td>Trp</td><td>Gin</td><td>Asn 125</td><td>Phe</td><td>Asp</td><td>Ser</td>

<td>Leu</td><td>Leu 130</td><td>Ile</td><td>Pro</td><td>Ala</td><td>Asp</td><td>His 135</td><td>Pro</td><td>ser</td><td>Arg</td><td>Lys</td><td>Lys 140</td><td>Gly</td><td>Asp</td><td>Asn</td><td>Tyr</td>

<td>Tyr 14 S</td><td>Leu</td><td>Asn</td><td>Arg</td><td>Thr</td><td>His 150</td><td>Met</td><td>Leu</td><td>Arg</td><td>Ala</td><td>His 155</td><td>Thr</td><td>Ser</td><td>Ala</td><td>His</td><td>Gin 160</td>

<td>Trp</td><td>Asp</td><td>Leu</td><td>Leu</td><td>His 16S</td><td>Ala</td><td>Gly</td><td>Leu</td><td>Asp</td><td>Ala 170</td><td>Phe</td><td>Leu</td><td>Val</td><td>Val</td><td>Gly 175</td><td>Asp</td>

<td>val</td><td>Tyr</td><td>Arg</td><td>Arg ISO</td><td>Asp</td><td>Gin</td><td>Ile</td><td>Asp</td><td>ser 185</td><td>Gin</td><td>His</td><td>Tyr</td><td>Pro</td><td>Ile 190</td><td>Phe</td><td>His</td>

<td>Gin</td><td>Leu</td><td>Glu 195</td><td>Ala</td><td>Val</td><td>Arg</td><td>Leu</td><td>Phe 200</td><td>Ser</td><td>Lys</td><td>His</td><td>Glu</td><td>Leu 205</td><td>Phe</td><td>Ala</td><td>Gly</td>

<td>ile</td><td>Lys 210</td><td>Asp</td><td>Gly</td><td>Glu</td><td>Ser</td><td>Leu 215</td><td>Gin</td><td>Leu</td><td>Phe</td><td>Glu</td><td>Gin 220</td><td>Ser</td><td>ser</td><td>Arg</td><td>Ser</td>

<td>Ala 225</td><td>His</td><td>Lys</td><td>Gin</td><td>Glu</td><td>Thr 230</td><td>His</td><td>Thr</td><td>Mec</td><td>Glu</td><td>Ala 23S</td><td>Val</td><td>Lys</td><td>Leu</td><td>Val</td><td>Glu 240</td>

<td>Phe</td><td>Asp</td><td>Leu</td><td>Lys</td><td>Gin 245</td><td>Thr</td><td>Leu</td><td>Thr</td><td>Arg</td><td>Leu 250</td><td>Met</td><td>Ala</td><td>His</td><td>Leu</td><td>Phe 255</td><td>Gly</td>

<td>Asp</td><td>Glu</td><td>Leu</td><td>Glu 260</td><td>Ile</td><td>Arg</td><td>Trp</td><td>Val</td><td>Aap 265</td><td>Cys</td><td>Tyr</td><td>Phe</td><td>Pro</td><td>Phe 270</td><td>Thr</td><td>His</td>

<td>Pro</td><td>Ser</td><td>Phe 275</td><td>Glu</td><td>Met</td><td>Glu</td><td>Ile</td><td>Asn 280</td><td>Phe</td><td>His</td><td>Gly</td><td>Glu</td><td>Trp 285</td><td>Leu</td><td>Glu</td><td>Val</td>

<td>Leu</td><td>Gly 290</td><td>Cys</td><td>Gly</td><td>Val</td><td>Met</td><td>Glu 295</td><td>Gin</td><td>Gin</td><td>Leu</td><td>val</td><td>Asn 300</td><td>Ser</td><td>Ala</td><td>Gly</td><td>Ala</td>

<td>Gin 305</td><td>ASp</td><td>Arg</td><td>Ile</td><td>Gly</td><td>Trp 310</td><td>Ala</td><td>Phe</td><td>Gly</td><td>Leu</td><td>Gly 315</td><td>Leu</td><td>Glu</td><td>Arg</td><td>Leu</td><td>Ala 320</td>

<td>Met</td><td>Ile</td><td>Leu</td><td>Tyr</td><td>Asp 325</td><td>ile</td><td>Pro</td><td>Aap</td><td>Ile</td><td>Arg 330</td><td>Leu</td><td>Phe</td><td>Trp</td><td>Cys</td><td>Glu 335</td><td>Asp</td>

<td>Glu</td><td>Arg</td><td>Phe</td><td>Leu 340</td><td>Lys</td><td>Gin</td><td>Phe</td><td>Cys</td><td>Val 345</td><td>Ser</td><td>Asn</td><td>Ile</td><td>Asn</td><td>Gin 350</td><td>Lys</td><td>Val</td>

<td>Lys</td><td>Phe</td><td>Gin 355</td><td>Pro</td><td>Leu</td><td>Ser</td><td>Lys</td><td>Tyr 360</td><td>Pro</td><td>Ala</td><td>Val</td><td>Ile</td><td>Asn 365</td><td>Asp</td><td>Ile</td><td>Ser</td>

<td>Phe</td><td>Trp 370</td><td>Leu</td><td>Pro</td><td>Ser</td><td>Glu</td><td>Asn 375</td><td>Tyr</td><td>Ala</td><td>Glu</td><td>Asn</td><td>Asp 380</td><td>Phe</td><td>Tyr</td><td>Asp</td><td>Leu</td>

<td>Val 385</td><td>Arg</td><td>Thr</td><td>Ile</td><td>Gly</td><td>Gly 390</td><td>Asp</td><td>Leu</td><td>Val</td><td>Glu</td><td>Lys 395</td><td>Val</td><td>Asp</td><td>Leu</td><td>Ile</td><td>Asp 400</td>

<td>Lys</td><td>Phe</td><td>Val</td><td>His</td><td>Pro 405</td><td>Lys</td><td>Thr</td><td>His</td><td>Lys</td><td>Thr 410</td><td>Ser</td><td>His</td><td>Cys</td><td>Tyr</td><td>Arg 415</td><td>Ile</td>

<td>Thr</td><td>Tyr</td><td>Arg</td><td>His 420</td><td>Met</td><td>Glu</td><td>Arg</td><td>Thr</td><td>Leu 425</td><td>ser</td><td>Gin</td><td>Arg</td><td>Glu</td><td>Val 430</td><td>Arg</td><td>His</td>

<td>Ile Gly</td><td>His Arg 450</td><td>Gin 435 Phe</td><td>Ala</td><td>Leu</td><td>Gin</td><td>Glu</td><td>Ala 440</td><td>Ala</td><td>Val</td><td>Gin</td><td>Leu</td><td>Leu 445</td><td>Gly</td><td>Val</td><td>Glu</td>

&lt;210&gt; 77 &lt;211&gt; 341 &lt;212&gt; PRT &lt;213&gt; Mycobacterium tuberculosis &lt;400&gt; 77

-146-

<td rowspan="2">Met 1</td><td rowspan="2">Leu</td><td colspan="13">Ser Pro Glu Ala Leu Thr Thr Ala Val Asp Ala Ala Gin</td><td rowspan="2">Gin</td>

<td colspan="3"> 5</td><td colspan="5"> 10</td><td colspan="5"> 15</td>

<td>Ala</td><td>Ile</td><td>Ala</td><td>Leu</td><td>Ala</td><td>Asp</td><td>Thr</td><td>Leu</td><td>Asp</td><td>Val</td><td>Leu</td><td>Ala</td><td>Arg</td><td>val</td><td>Lys</td><td>Thr</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Glu</td><td>His</td><td>Leu</td><td>Gly</td><td>Asp</td><td>Arg</td><td>Ser</td><td>Pro</td><td>Leu</td><td>Ala</td><td>Leu</td><td>Ala</td><td>Arg</td><td>Gin</td><td>Ala</td><td>Leu</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Ala</td><td>Val</td><td>Leu</td><td>Pro</td><td>Lys</td><td>Glu</td><td>Gin</td><td>Arg</td><td>Ala</td><td>Glu</td><td>Ala</td><td>Gly</td><td>Lys</td><td>Arg</td><td>Val</td><td>Asn</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Ala</td><td>Ala</td><td>Arg</td><td>Asn</td><td>Ala</td><td>Ala</td><td>Gin</td><td>Arg</td><td>Ser</td><td>Tyr</td><td>Asp</td><td>Glu</td><td>Arg</td><td>Leu</td><td>Ala</td><td>Thr</td>

<td> £5</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Leu</td><td>Arg</td><td>Ala</td><td>Glu</td><td>Arg</td><td>Asp</td><td>Ala</td><td>Ala</td><td>Val</td><td>Leu</td><td>Val</td><td>Ala</td><td>Glu</td><td>Gly</td><td>Ile</td><td>Asp</td>

<td></td><td></td><td></td><td></td><td>Θ5</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Val</td><td>Thr</td><td>Leu</td><td>Pro</td><td>Ser</td><td>Thr</td><td>Arg</td><td>Val</td><td>Pro</td><td>Ala</td><td>Gly</td><td>Ala</td><td>Arg</td><td>His</td><td>Pro</td><td>Ile</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Ile</td><td>Met</td><td>Leu</td><td>Ala</td><td>Glu</td><td>His</td><td>Val</td><td>Ala</td><td>Asp</td><td>Thr</td><td>Phe</td><td>Ile</td><td>Ala</td><td>Met</td><td>Gly</td><td>Trp</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Glu</td><td>Leu</td><td>Ala</td><td>Glu</td><td>Gly</td><td>Pro</td><td>Glu</td><td>val</td><td>Glu</td><td>Thr</td><td>Glu</td><td>Gin</td><td>Phe</td><td>Asn</td><td>Phe</td><td>Asp</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Ala</td><td>Leu</td><td>Asn</td><td>Phe</td><td>Pro</td><td>Ala</td><td>Asp</td><td>His</td><td>Pro</td><td>Ala</td><td>Arg</td><td>Gly</td><td>Glu</td><td>Gin</td><td>Asp</td><td>Thr</td>

<td> 14 5</td><td></td><td></td><td></td><td></td><td>ISO</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Phe</td><td>Tyr</td><td>Ile</td><td>Ala</td><td>Pro</td><td>Glu</td><td>Asp</td><td>Ser</td><td>Arg</td><td>Gin</td><td>Leu</td><td>Leu</td><td>Arg</td><td>Thr</td><td>His</td><td>Thr</td>

<td></td><td></td><td></td><td></td><td>16S</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Ser</td><td>Pro</td><td>val</td><td>Gin</td><td>Ile</td><td>Arg</td><td>Thr</td><td>Leu</td><td>Leu</td><td>Ala</td><td>Arg</td><td>Glu</td><td>Leu</td><td>Pro</td><td>Val</td><td>Tyr</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>ile</td><td>Ile</td><td>Ser</td><td>Ile</td><td>Gly</td><td>Arg</td><td>Thr</td><td>Phe</td><td>Arg</td><td>Thr</td><td>Asp</td><td>Glu</td><td>Leu</td><td>Asp</td><td>Ala</td><td>Thr</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>His</td><td>Thr</td><td>Pro</td><td>Ile</td><td>Phe</td><td>His</td><td>Gin</td><td>Val</td><td>Glu</td><td>Gly</td><td>Leu</td><td>Ala</td><td>Val</td><td>Asp</td><td>Arg</td><td>Gly</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Leu</td><td>Ser</td><td>Met</td><td>Ala</td><td>His</td><td>Leu</td><td>Arg</td><td>Gly</td><td>Thr</td><td>Leu</td><td>Asp</td><td>Ala</td><td>Phe</td><td>Ala</td><td>Arg</td><td>Ala</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>Glu</td><td>Phe</td><td>Gly</td><td>Pro</td><td>Ser</td><td>Ala</td><td>Arg</td><td>Thr</td><td>Arg</td><td>Ile</td><td>Arg</td><td>Pro</td><td>His</td><td>Phe</td><td>Phe</td><td>Pro</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

<td>Phe</td><td>Thr</td><td>Glu</td><td>Pro</td><td>Ser</td><td>Ala</td><td>Glu</td><td>Val</td><td>A3p</td><td>Val</td><td>Trp</td><td>Phe</td><td>Ala</td><td>Asn</td><td>Lye</td><td>Ile</td>

<td></td><td></td><td></td><td>2S0</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>Gly</td><td>Gly</td><td>Ala</td><td>Ala</td><td>Trp</td><td>Val</td><td>Glu</td><td>Trp</td><td>Gly</td><td>Gly</td><td>Cys</td><td>Gly</td><td>Met</td><td>Val</td><td>His</td><td>Pro</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>Asn</td><td>Val</td><td>Leu</td><td>Arg</td><td>Ala</td><td>Thr</td><td>Gly</td><td>Ile</td><td>AŚp</td><td>Pro</td><td>Asp</td><td>Leu</td><td>Tyr</td><td>Ser</td><td>Gly</td><td>Phe</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>Ala</td><td>Phe</td><td>Gly</td><td>Met</td><td>Gly</td><td>Leu</td><td>Glu</td><td>Arg</td><td>Thr</td><td>Leu</td><td>Gin</td><td>Phe</td><td>Arg</td><td>Asn</td><td>Gly</td><td>Ile</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td>pro</td>

Phe Gly Val Gly Ala 340 &lt;210&gt; 78 &lt;211&gt; 458 &lt;212&gt; PRT &lt;213&gt; Caenorhabditis elegans &lt;400&gt; 78

-147-

<td rowspan="2">Met 1</td><td rowspan="2">Arg</td><td rowspan="2">Phe</td><td rowspan="2">Leu</td><td colspan="2" rowspan="2">Ser Thr 5</td><td rowspan="2">Tyr</td><td rowspan="2">Gin</td><td colspan="7">Thr Gly Ala Arg Arg Cys Cys</td><td rowspan="2">Thr</td>

<td colspan="2"> 10</td><td colspan="5"> 15</td>

<td>Met</td><td>Leu</td><td>Phe</td><td>Gin</td><td>Lys</td><td>Asn</td><td>Ala</td><td>Lys</td><td>Ser</td><td>Ile</td><td>Arg</td><td>Ala</td><td>Thr</td><td>Leu</td><td>Met</td><td>Ser</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Thr</td><td>Thr</td><td>Ala</td><td>Ala</td><td>Ala</td><td>Glu</td><td>Glu</td><td>Ser</td><td>Arg</td><td>Lys</td><td>Ile</td><td>Val</td><td>Lys</td><td>Glu</td><td>Glu</td><td>Val</td>

<td></td><td></td><td>3S</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Phe</td><td>Glu</td><td>Leu</td><td>Asp</td><td>Gly</td><td>Arg</td><td>Lys</td><td>Tyr</td><td>Thr</td><td>Pro</td><td>Asp</td><td>Ala</td><td>Leu</td><td>Tyr</td><td>Asn</td><td>Leu</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Ser</td><td>Pro</td><td>Gly</td><td>val</td><td>Arg</td><td>Arg</td><td>Leu</td><td>Leu</td><td>Asp</td><td>Arg</td><td>Arg</td><td>Ile</td><td>Leu</td><td>Gin</td><td>Glu</td><td>Ser</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Ser</td><td>Asn</td><td>Pro</td><td>Leu</td><td>Asn</td><td>Leu</td><td>Leu</td><td>Lys</td><td>Arg</td><td>Arg</td><td>Ile</td><td>val</td><td>Asp</td><td>Tyr</td><td>Val</td><td>His</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Gin</td><td>Thr</td><td>Tyr</td><td>Arg</td><td>Lys</td><td>Pro</td><td>Gly</td><td>Asn</td><td>Arg</td><td>Ser</td><td>Pro</td><td>Leu</td><td>Phe</td><td>Thr</td><td>Ile</td><td>Cys</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Glu</td><td>Ser</td><td>Glu</td><td>Pro</td><td>Arg</td><td>Val</td><td>Val</td><td>Thr</td><td>Thr</td><td>Tyr</td><td>Gin</td><td>Asn</td><td>Phe</td><td>Asp</td><td>Ser</td><td>Leu</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Leu</td><td>Thr</td><td>Pro</td><td>Glu</td><td>Asp</td><td>His</td><td>Val</td><td>Ser</td><td>Arg</td><td>Arg</td><td>Pro</td><td>Ser</td><td>Asp</td><td>Thr</td><td>Tyr</td><td>Tyr</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Val</td><td>Asn</td><td>His</td><td>Glu</td><td>His</td><td>Cya</td><td>Leu</td><td>Arg</td><td>Ala</td><td>His</td><td>Thr</td><td>Ser</td><td>Ala</td><td>His</td><td>Gin</td><td>His</td>

<td> 145</td><td></td><td></td><td></td><td></td><td>ISO</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Aan</td><td>Leu</td><td>Met</td><td>Gin</td><td>Ser</td><td>Gly</td><td>Leu</td><td>Asp</td><td>Ala</td><td>Phe</td><td>Leu</td><td>val</td><td>ile</td><td>Gly</td><td>Asp</td><td>Val</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Tyr</td><td>Arg</td><td>Arg</td><td>Asp</td><td>Glu</td><td>Val</td><td>Asp</td><td>Arg</td><td>Thr</td><td>His</td><td>Tyr</td><td>Pro</td><td>Cys</td><td>Phe</td><td>His</td><td>Gin</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Ile</td><td>Glu</td><td>Gly</td><td>Val</td><td>Arg</td><td>Leu</td><td>Tyr</td><td>Ser</td><td>Lys</td><td>Asp</td><td>Glu</td><td>Leu</td><td>Leu</td><td>Gly</td><td>Lys</td><td>Lys</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Pro</td><td>Asp</td><td>Gly</td><td>Lys</td><td>Asn</td><td>Val</td><td>Ala</td><td>Glu</td><td>Leu</td><td>Phe</td><td>Ser</td><td>Thr</td><td>Ala</td><td>Ala</td><td>Ser</td><td>Ala</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Ala</td><td>Thr</td><td>Glu</td><td>Arg</td><td>Ser</td><td>Pro</td><td>Glu</td><td>Lys</td><td>Gin</td><td>Glu</td><td>Lys</td><td>His</td><td>Thr</td><td>Leu</td><td>Asp</td><td>Ala</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>Thr</td><td>Lys</td><td>Ala</td><td>Ala</td><td>Glu</td><td>Ile</td><td>Gin</td><td>Leu</td><td>Lys</td><td>Gin</td><td>Phe</td><td>Leu</td><td>Glu</td><td>Asn</td><td>Leu</td><td>Cys</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

<td>Asp</td><td>Glu</td><td>Leu</td><td>Phe</td><td>Gly</td><td>Ly3</td><td>Asp</td><td>Ala</td><td>Glu</td><td>Lys</td><td>Arg</td><td>Trp</td><td>Val</td><td>Asp</td><td>Ala</td><td>Tyr</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td>26S</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>Phe</td><td>Pro</td><td>Phe</td><td>Thr</td><td>His</td><td>Pro</td><td>Ser</td><td>Trp</td><td>□ lu</td><td>Leu</td><td>Glu</td><td>Val</td><td>Phe</td><td>Tyr</td><td>Asn</td><td>Gly</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>Gin</td><td>Trp</td><td>Leu</td><td>Glu</td><td>val</td><td>Leu</td><td>Gly</td><td>Cys</td><td>Gly</td><td>Ile</td><td>Met</td><td>Glu</td><td>Gin</td><td>Lys</td><td>Leu</td><td>Leu</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>Glu</td><td>Ser</td><td>Ala</td><td>Gly</td><td>Val</td><td>Thr</td><td>Asp</td><td>Lys</td><td>Ile</td><td>Gly</td><td>Trp</td><td>Ala</td><td>Phe</td><td>Gly</td><td>Ile</td><td>Gly</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

<td>Leu</td><td>Glu</td><td>Arg</td><td>Ile</td><td>Ala</td><td>Met</td><td>Val</td><td>Leu</td><td>Tyr</td><td>Gly</td><td>Ile</td><td>Pro</td><td>Asp</td><td>Ile</td><td>Arg</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td>

<td>Phe</td><td>Trp</td><td>Ser</td><td>Lys</td><td>Asp</td><td>Thr</td><td>Gly</td><td>Phe</td><td>Leu</td><td>Ser</td><td>Gin</td><td>Phe</td><td>Ala</td><td>Gly</td><td>Lys</td><td>Met</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td>

<td>Pro</td><td>Gly</td><td>Glu</td><td>Asp</td><td>Val</td><td>Lys</td><td>Tyr</td><td>Lys</td><td>Gin</td><td>ile</td><td>Ser</td><td>Ala</td><td>His</td><td>Pro</td><td>Gin</td><td>Val</td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td>

<td>ile</td><td>Phe</td><td>Asp</td><td>Ile</td><td>ser</td><td>Phe</td><td>Phe</td><td>Leu</td><td>Pro</td><td>Ser</td><td>Thr</td><td>Val</td><td>Gin</td><td>Phe</td><td>Asn</td><td>Asp</td>

<td></td><td> 370</td><td></td><td></td><td></td><td></td><td>37S</td><td></td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td>

<td>Ket</td><td>Thr</td><td>Ser</td><td>Asp</td><td>Val</td><td>Tyr</td><td>Asp</td><td>Thl*</td><td>Ile</td><td>Arg</td><td>Thr</td><td>Val</td><td>Gly</td><td>Gly</td><td>Glu</td><td>Leu</td>

<td> 385</td><td></td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td>

<td>Val</td><td>Glu</td><td>Gin</td><td>Val</td><td>Lys</td><td>Leu</td><td>Thr</td><td>Asp</td><td>Glu</td><td>Phe</td><td>Glu</td><td>Asn</td><td>Lys</td><td>Lys</td><td>Lys</td><td>Glu</td>

<td></td><td></td><td></td><td></td><td> 405</td><td></td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td>

<td>Lys</td><td>Lys</td><td>Ser</td><td>Gin</td><td>Thr</td><td>Tyr</td><td>Arg</td><td>Ile</td><td>Val</td><td>Tyr</td><td>Arg</td><td>Ser</td><td>His</td><td>Glu</td><td>Arg</td><td>Ala</td>

<td></td><td></td><td></td><td> 420</td><td></td><td></td><td></td><td></td><td> 425</td><td></td><td></td><td></td><td></td><td> 430</td><td></td><td></td>

<td>Leu</td><td>Thr</td><td>Lys</td><td>Glu</td><td>Glu</td><td>Val</td><td>Asn</td><td>Val</td><td>Ile</td><td>His</td><td>Lys</td><td>Gin</td><td>ile</td><td>Glu</td><td>Gin</td><td>Ser</td>

<td></td><td></td><td> 435</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td></td><td></td><td></td>

<td>Leu</td><td>Ala</td><td>Ser</td><td>Ser</td><td>Phe</td><td>Gly</td><td>Val</td><td>Thr</td><td>Leu</td><td>Arg</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>4S0</td><td></td><td></td><td></td><td></td><td> 455</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 79 &lt;211&gt; 469 &lt;212&gt; PRT &lt;213&gt; Saccharomyces cerevisiae &lt;400&gt; 79

-148-

<td>Met 1</td><td>Phe</td><td>Leu</td><td>Asn</td><td>Arg 5</td><td>Mec</td><td>Met</td><td>Lys</td><td>Thr</td><td>Arg 10</td><td>Thr</td><td>Gly</td><td>Leu</td><td>Tyr</td><td>Arg 15</td><td>LeU</td>

<td>Tyr</td><td>Ser</td><td>Thr</td><td>Leu 20</td><td>Lys</td><td>Val</td><td>Pro</td><td>His</td><td>Val 25</td><td>Glu</td><td>Ile</td><td>Asn</td><td>Gly</td><td>Ile 30</td><td>Lys</td><td>Tyr</td>

<td>Lys</td><td>Thr</td><td>Asp 35</td><td>Pro</td><td>Gln</td><td>Thr</td><td>Thr</td><td>Asn 40</td><td>Val</td><td>Thr</td><td>Asp</td><td>Ser</td><td>Ile 45</td><td>Ile</td><td>Lys</td><td>Leu</td>

<td>Thr</td><td>Asp 50</td><td>Arg</td><td>Ser</td><td>Leu</td><td>His</td><td>Leu 55</td><td>Lys</td><td>Glu</td><td>Ser</td><td>His</td><td>Pro 60</td><td>Val</td><td>Gly</td><td>Ile</td><td>Leu</td>

<td>Arg 65</td><td>Asp</td><td>Leu</td><td>Ile</td><td>Glu</td><td>Lys 70</td><td>Lys</td><td>Leu</td><td>Asn</td><td>Ser</td><td>Val 75</td><td>A3p</td><td>Asn</td><td>Thr</td><td>Phe</td><td>Lys 80</td>

<td>Ile</td><td>Phe</td><td>Asn</td><td>Asn</td><td>Phe 85</td><td>Lys</td><td>Pro</td><td>Val</td><td>Val</td><td>Thr 90</td><td>Thr</td><td>Met</td><td>Glu</td><td>Asn</td><td>Phe 95</td><td>Asp</td>

<td>Ser</td><td>Leu</td><td>Gly</td><td>Phe 100</td><td>Pro</td><td>Lys</td><td>Asp</td><td>His</td><td>Pro 105</td><td>Gly</td><td>Arg</td><td>Ser</td><td>Lys</td><td>Ser 110</td><td>Asp</td><td>Thr</td>

<td>Tyr</td><td>Tyr</td><td>Ile 115</td><td>Asn</td><td>Glu</td><td>Thr</td><td>His</td><td>Leu 120</td><td>Leu</td><td>Arg</td><td>Thr</td><td>His</td><td>Thr 125</td><td>ser</td><td>Ala</td><td>His</td>

<td>Glu</td><td>Leu 130</td><td>Glu</td><td>Cys</td><td>phe</td><td>Gln</td><td>Lys 135</td><td>ile</td><td>Arg</td><td>Asn</td><td>Asp</td><td>Ser 140</td><td>Asp</td><td>Asn</td><td>Ile</td><td>Lys</td>

<td>Ser 145</td><td>Gly</td><td>Phe</td><td>Leu</td><td>Ile</td><td>Ser 150</td><td>Ala</td><td>Asp</td><td>Val</td><td>Tyr</td><td>Arg 155</td><td>Arg</td><td>ASp</td><td>Glu</td><td>Ile</td><td>Aap 160</td>

<td>Lys</td><td>Thr</td><td>His</td><td>Tyr</td><td>Pro 165</td><td>Val</td><td>Phe</td><td>ΗΪ3</td><td>Gln</td><td>Met 170</td><td>Glu</td><td>Gly</td><td>Ala</td><td>Thr</td><td>ile 175</td><td>Trp</td>

<td>Lys</td><td>Arg</td><td>Thr</td><td>Lys 180</td><td>Ala</td><td>Asp</td><td>Val</td><td>Gly</td><td>Val 185</td><td>Lys</td><td>Glu</td><td>Pro</td><td>Mec</td><td>Tyr 130</td><td>ile</td><td>Glu</td>

<td>Lys</td><td>Ile</td><td>Arg 195</td><td>Glu</td><td>Asp</td><td>Ile</td><td>Arg</td><td>Gln 200</td><td>Val</td><td>Glu</td><td>Asn</td><td>Leu</td><td>Leu 205</td><td>Asn</td><td>Lys</td><td>Glu</td>

<td>Asn</td><td>Val 210</td><td>Lys</td><td>Ile</td><td>Thr</td><td>Val</td><td>Asp 215</td><td>Asp</td><td>Asp</td><td>Thr</td><td>Ile</td><td>Pro 220</td><td>Leu</td><td>Lys</td><td>Glu</td><td>Asn</td>

<td>Asn 225</td><td>Pro</td><td>Lys</td><td>Gln</td><td>Glu</td><td>Tyr 230</td><td>Mec</td><td>Ser</td><td>Asp</td><td>Leu</td><td>Glu 235</td><td>Val</td><td>Asp</td><td>Leu</td><td>Cys</td><td>Ser 240</td>

<td>Gln</td><td>His</td><td>Leu</td><td>Lys</td><td>Arg 245</td><td>Ser</td><td>Ile</td><td>Glu</td><td>Leu</td><td>Ile 250</td><td>Val</td><td>Ser</td><td>Glu</td><td>Val</td><td>Phe 255</td><td>Asn</td>

<td>Lys</td><td>Lys</td><td>Ile</td><td>Ser 260</td><td>Ser</td><td>Met</td><td>Ile</td><td>Lys</td><td>Asn 265</td><td>Lys</td><td>Ala</td><td>Asn</td><td>Asn</td><td>Thr 270</td><td>pro</td><td>Lys</td>

<td>Glu</td><td>Leu</td><td>Lys</td><td>val</td><td>Arg</td><td>Trp</td><td>ile</td><td>Asn</td><td>Ala</td><td>Tyr</td><td>Phe</td><td>Pro</td><td>Trp</td><td>Thr</td><td>Ala</td><td>Pro</td>

275 280 285

Ser Trp Glu Ile Glu Val Trp Trp Gln Gly Glu Trp Leu Glu Leu cya 290 295 300

<td colspan="2">Gly Cys Gly Leu Ile Arg Gln Asp Val</td><td>Leu Leu Arg</td><td>Ala Gly Tyr Lys</td>

<td> 305</td><td> 310</td><td> 315</td><td> 320</td>

Pro Ser Glu Thr Ile Gly Trp Ala Phe Gly Leu Gly Leu Asp Arg Ile 325 330 335

Ala Met Leu Leu Phe Glu Ile Pro Asp Ile Arg Leu Leu Trp Ser Arg 340 345 3S0

Asp Glu Arg Phe Ser Arg Gln Phe Ser Lys Gly Leu Ile Thr Ser Phe 355 360 365

<td rowspan="2">Lys</td><td colspan="5" rowspan="2">Pro Tyr Ser Lys His 370</td><td colspan="9">Pro Gly Ser Phe Arg Asp Val Ala Phe</td><td rowspan="2">Trp</td>

<td colspan="5"> 375</td><td colspan="4"> 380</td>

<td>Leu</td><td>Pro</td><td>Glu</td><td>Asp</td><td>Lys</td><td>Pro</td><td>Asp</td><td>Ile</td><td>His</td><td>Gln</td><td>val</td><td>His</td><td>Glu</td><td>Asn</td><td>Asp</td><td>Leu</td>

<td> 385</td><td></td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td>

<td>Met</td><td>Glu</td><td>Ile</td><td>Ile</td><td>Arg</td><td>Asn</td><td>Ile</td><td>Ala</td><td>Gly</td><td>Asp</td><td>Leu</td><td>Val</td><td>Glu</td><td>Ser</td><td>Val</td><td>Lys</td>

<td></td><td></td><td></td><td></td><td> 405</td><td></td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td>

<td>Leu</td><td>Val</td><td>Asp</td><td>Ser</td><td>Phe</td><td>Thr</td><td>His</td><td>Pro</td><td>Lys</td><td>Thr</td><td>Gly</td><td>Arg</td><td>Lys</td><td>Ser</td><td>Met</td><td>Cya</td>

<td></td><td></td><td></td><td> 420</td><td></td><td></td><td></td><td></td><td> 425</td><td></td><td></td><td></td><td></td><td> 430</td><td></td><td></td>

<td>Tyr</td><td>Arg</td><td>Ile</td><td>Asn</td><td>Tyr</td><td>Gln</td><td>Ser</td><td>Met</td><td>Asp</td><td>Arg</td><td>Asn</td><td>Leu</td><td>Thr</td><td>Asn</td><td>Ala</td><td>Glu</td>

<td></td><td></td><td> 435</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td></td><td></td><td></td>

<td>val</td><td>Asn</td><td>Thr</td><td>Leu</td><td>Gln</td><td>Asp</td><td>Met</td><td>Val</td><td>Cys</td><td>Ser</td><td>Lys</td><td>Leu</td><td>val</td><td>Lys</td><td>Glu</td><td>Tyr</td>

<td></td><td> 450</td><td></td><td></td><td></td><td></td><td> 4 55</td><td></td><td></td><td></td><td></td><td> 460</td><td></td><td></td><td></td><td></td>

<td>Ser</td><td>Val</td><td>Glu</td><td>Leu</td><td>Arg</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>46S</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 80 &lt;211&gt; 499 &lt;212&gt; PRT &lt;213&gt; Schizosaccharomyces pombe &lt;400&gt; 80

-149-

<td>Met 1</td><td colspan="2">Ser Lys</td><td>Leu</td><td colspan="2">Glu Ala 5</td><td>Leu</td><td>Gin</td><td>Val</td><td>Phe 10</td><td colspan="4">Leu Leu Glu Lys</td><td>Leu 15</td><td>Asn</td>

<td>Glu</td><td>Lys</td><td>Asn</td><td>Glu</td><td>Ile</td><td>Pro</td><td>Asn</td><td>Thr</td><td>Ser</td><td>His</td><td>Leu</td><td>Glu</td><td>Phe</td><td>Asp</td><td>Gly</td><td>Lys</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td>2S</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Lys</td><td>Leu</td><td>Gly</td><td>Pro</td><td>Gin</td><td>Glu</td><td>Ala</td><td>Gin</td><td>Ser</td><td>Ala</td><td>ile</td><td>Leu</td><td>ser</td><td>Leu</td><td>Ala</td><td>Ala</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Lys</td><td>Asn</td><td>Met</td><td>Ile</td><td>Glu</td><td>Phe</td><td>Ser</td><td>Arg</td><td>His</td><td>Glu</td><td>Ile</td><td>Glu</td><td>Ile</td><td>Tyr</td><td>Asn</td><td>Leu</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Thr</td><td>Ala</td><td>Glu</td><td>Gly</td><td>Glu</td><td>Asn</td><td>ile</td><td>Cys</td><td>Ala</td><td>Asn</td><td>Gly</td><td>Ser</td><td>His</td><td>Glu</td><td>Ala</td><td>Lys</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Val</td><td>Tyr</td><td>Asn</td><td>Glu</td><td>Ile</td><td>Cys</td><td>Ala</td><td>Ser</td><td>Met</td><td>Ser</td><td>Gly</td><td>Leu</td><td>Asn</td><td>Ile</td><td>Gly</td><td>Glu</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Leu</td><td>Lys</td><td>Lys</td><td>Lys</td><td>Leu</td><td>Gly</td><td>Asn</td><td>Ser</td><td>Ala</td><td>Gly</td><td>Ile</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Arg</td><td>Ala</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Phe</td><td>Lys</td><td>Leu</td><td>Gly</td><td>Trp</td><td>Ile</td><td>Lys</td><td>Lys</td><td>Asp</td><td>Gly</td><td>Asp</td><td>Lys</td><td>Leu</td><td>Val</td><td>Lys</td><td>Asn</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Thr</td><td>Asp</td><td>Ser</td><td>Ile</td><td>Thr</td><td>Asp</td><td>Glu</td><td>Thr</td><td>Pro</td><td>Lys</td><td>Val</td><td>Leu</td><td>Ser</td><td>Glu</td><td>Ile</td><td>Lys</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Glu</td><td>His</td><td>Gly</td><td>Thr</td><td>Ile</td><td>Ser</td><td>ASp</td><td>Ser</td><td>Lys</td><td>Thr</td><td>Leu</td><td>Thr</td><td>Asp</td><td>Leu</td><td>Lys</td><td>Lys</td>

<td> 14 5</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Arg</td><td>Lys</td><td>Leu</td><td>Val</td><td>Glu</td><td>Arg</td><td>Asn</td><td>Lys</td><td>Ile</td><td>Mec</td><td>Tyr</td><td>Phe</td><td>Ser</td><td>Leu</td><td>Arg</td><td>Lys</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Gly</td><td>Pro</td><td>Asn</td><td>Phe</td><td>Ser</td><td>Leu</td><td>Gin</td><td>Ile</td><td>Glu</td><td>Lys</td><td>Leu</td><td>Asn</td><td>Thr</td><td>Asp</td><td>Leu</td><td>Thr</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Ala</td><td>Glu</td><td>Met</td><td>Ile</td><td>Thr</td><td>Ser</td><td>Arg</td><td>Ser</td><td>Trp</td><td>GlU</td><td>Ser</td><td>Ala</td><td>Lys</td><td>Phe</td><td>Lys</td><td>Ser</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Tyr</td><td>Asn</td><td>Phe</td><td>Ala</td><td>Ala</td><td>Glu</td><td>Gly</td><td>Ile</td><td>pro</td><td>Pro</td><td>Ala</td><td>Gly</td><td>Gly</td><td>Cys</td><td>Leu</td><td>His</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Pro</td><td>Leu</td><td>Met</td><td>Lys</td><td>Val</td><td>Arg</td><td>Glu</td><td>Glu</td><td>Phe</td><td>Arg</td><td>Lys</td><td>Phe</td><td>Phe</td><td>Phe</td><td>Glu</td><td>Leu</td>

<td>225 Gly</td><td>Phe</td><td>Glu</td><td>Glu</td><td>Met.</td><td>230 Pro</td><td>Thr</td><td>Asn</td><td>Asn</td><td>Phe</td><td>235 Val</td><td>Glu</td><td>Ser</td><td>Gly</td><td>Phe</td><td>240 Trp</td>

<td>Asn</td><td>Phe</td><td>Asp</td><td>Ala</td><td>24 5 Leu</td><td>Phe</td><td>Val</td><td>Pro</td><td>Gin</td><td>250 Gin</td><td>Kas</td><td>Ser</td><td>Ala</td><td>Arg</td><td>255 Asp</td><td>Ala</td>

<td>Gin</td><td>Asp</td><td>Thr</td><td>260 Phe</td><td>Phe</td><td>Leu</td><td>Lys</td><td>Val</td><td>265 Pro</td><td>Ala</td><td>Ser</td><td>Thr</td><td>Asp</td><td>270 Lys</td><td>Leu</td><td>Pro</td>

<td>Asp</td><td>Pro</td><td>275 GlU</td><td>Tyr</td><td>Val</td><td>Ala</td><td>Arg</td><td>280 Val</td><td>Lys</td><td>Ala</td><td>Thr</td><td>His</td><td>285 Glu</td><td>Asn</td><td>Gly</td><td>Gly</td>

<td>Glu</td><td>290 Thr</td><td>Lys</td><td>Gly</td><td>Ile</td><td>Gly</td><td>295 Tyr</td><td>Arg</td><td>Ala</td><td>Pro</td><td>Phe</td><td>300 Ser</td><td>Leu</td><td>Glu</td><td>Glu</td><td>Thr</td>

<td>305 Arg</td><td>Lys</td><td>Leu</td><td>Val</td><td>Leu</td><td>310 Arg</td><td>Thr</td><td>His</td><td>Thr</td><td>Thr</td><td>315 Ala</td><td>Val</td><td>Ser</td><td>Ala</td><td>Asn</td><td>320 Met</td>

<td>Leu</td><td>Tyr</td><td>Lys</td><td>Leu</td><td>325 Ala</td><td>Gin</td><td>Asn</td><td>Gly</td><td>Phe</td><td>330 His</td><td>Pro</td><td>Ala</td><td>Lys</td><td>Tyr</td><td>335 Phe</td><td>Ser</td>

<td>Ile</td><td>Asp</td><td>Arg</td><td>340 Val</td><td>Phe</td><td>Arg</td><td>Asn</td><td>Glu</td><td>345 Thr</td><td>Val</td><td>Asp</td><td>Ala</td><td>Thr</td><td>350 His</td><td>Leu</td><td>Ala</td>

<td>Glu</td><td>Phe</td><td>355 His</td><td>Gin</td><td>Val</td><td>Glu</td><td>Gly</td><td>360 Val</td><td>Ile</td><td>Cys</td><td>Asp</td><td>Arg</td><td>365 Asn</td><td>Ile</td><td>Thr</td><td>Leu</td>

<td>Gly</td><td>370 Asp</td><td>Leu</td><td>Ile</td><td>Gly</td><td>Phe</td><td>375 Leu</td><td>Glu</td><td>Val</td><td>Phe</td><td>Phe</td><td>380 Gly</td><td>Lys</td><td>Met</td><td>Asn</td><td>Val</td>

<td>385 Lys</td><td>Asn</td><td>Leu</td><td>Arg</td><td>Phe</td><td>390 Lys</td><td>Pro</td><td>Ala</td><td>Tyr</td><td>Asn</td><td>395 Pro</td><td>Tyr</td><td>Thr</td><td>Glu</td><td>Pro</td><td>400 Ser</td>

<td>Leu</td><td>Glu</td><td>Val</td><td>Phe</td><td>405 Ser</td><td>Tyr</td><td>His</td><td>Glu</td><td>Lys</td><td>410 Leu</td><td>Gly</td><td>Lys</td><td>Trp</td><td>val</td><td>415 Glu</td><td>Val</td>

<td>Gly</td><td>Asn</td><td>Ser</td><td>420 Gly</td><td>Met</td><td>Phe</td><td>Arg</td><td>Pro</td><td>425 Glu</td><td>Met</td><td>Leu</td><td>Glu</td><td>Pro</td><td>430 Met</td><td>Gly</td><td>Leu</td>

<td>Pro</td><td>Lys</td><td>435 Asp</td><td>Val</td><td>Arg</td><td>Cys</td><td>Leu</td><td>440 Gly</td><td>Phe</td><td>Gly</td><td>Leu</td><td>Ser</td><td>445 Leu</td><td>Glu</td><td>Arg</td><td>Pro</td>

<td>Thr</td><td>450 Met</td><td>Ile</td><td>Lys</td><td>Tyr</td><td>Gly</td><td>4 55 Val</td><td>Ala</td><td>Asp</td><td>Ile</td><td>Arg</td><td>460 Gin</td><td>Leu</td><td>Ile</td><td>Gly</td><td>Pro</td>

<td>4 55 Lys</td><td>Val</td><td>Asn</td><td>Leu</td><td>Asp</td><td>470 Leu</td><td>ile</td><td>Glu</td><td>Ala</td><td>Ser</td><td>475 Pro</td><td>Ala</td><td>Val</td><td>Arg</td><td>Leu</td><td>480 Asp</td>

<td>Lys</td><td>Glu</td><td>Glu</td><td></td><td> 485</td><td></td><td></td><td></td><td></td><td> 490</td><td></td><td></td><td></td><td></td><td> 4 95</td><td></td>

&lt;210&gt; 81 &lt;211&gt; 338 &lt;212&gt; PRT &lt;213&gt; Pseudomonas aeruginosa

-150&lt;400&gt;81

<td>Met 1</td><td>Glu</td><td>Asn</td><td>Leu</td><td>Asp 5</td><td>Ala</td><td>Leu</td><td>Val</td><td>Ser</td><td>Gin 10</td><td>Ala</td><td>Leu</td><td>Glu</td><td>Ala</td><td>Val 15</td><td>Arg</td>

<td>His</td><td>Thr</td><td>Glu</td><td>Asp 20</td><td>Val</td><td>Asn</td><td>Ala</td><td>Leu</td><td>Glu 25</td><td>Gin</td><td>Ile</td><td>Arg</td><td>Val</td><td>His 30</td><td>Tyr</td><td>Leu</td>

<td>Gly</td><td>Lys</td><td>Lys 35</td><td>Gly</td><td>Glu</td><td>Leu</td><td>Thr</td><td>Gin 40</td><td>Val</td><td>Met</td><td>Lys</td><td>Thr</td><td>Leu 45</td><td>Gly</td><td>Asp</td><td>Leu</td>

<td>Pro</td><td>Ala SO</td><td>Glu</td><td>Glu</td><td>Arg</td><td>Pro</td><td>Lys 55</td><td>Val</td><td>Gly</td><td>Ala</td><td>Leu</td><td>Ile 60</td><td>Asn</td><td>Val</td><td>Ala</td><td>Lys</td>

<td>Glu 65</td><td>Lys</td><td>Val</td><td>Gin</td><td>Asp</td><td>Val 70</td><td>Leu</td><td>Asn</td><td>Ala</td><td>Arg</td><td>Lys 75</td><td>Thr</td><td>Glu</td><td>Leu</td><td>Glu</td><td>Gly 80</td>

<td>Ala</td><td>Ala</td><td>Leu</td><td>Ala</td><td>Ala 85</td><td>Arg</td><td>Leu</td><td>Ala</td><td>Ala</td><td>Glu 90</td><td>Arg</td><td>Ile</td><td>ASp</td><td>Val</td><td>Thr 95</td><td>Len</td>

<td>Pro</td><td>Gly</td><td>Arg</td><td>Gly 100</td><td>Gin</td><td>Leu</td><td>Ser</td><td>Gly</td><td>Gly 105</td><td>Leu</td><td>Kis</td><td>Pro</td><td>val</td><td>Thr 110</td><td>Arg</td><td>Thr</td>

<td>Leu</td><td>Glu</td><td>Arg 115</td><td>Tle</td><td>Glu</td><td>Gin</td><td>Cys</td><td>Phe 120</td><td>Ser</td><td>Arg</td><td>Ile</td><td>Gly</td><td>Tyr 125</td><td>Glu</td><td>Val</td><td>Ala</td>

<td>Glu</td><td>Gly 13 0</td><td>Pro</td><td>Glu</td><td>Val</td><td>Glu</td><td>Asp 135</td><td>Asp</td><td>Tyr</td><td>His</td><td>Asn</td><td>Phe 140</td><td>Glu</td><td>Ala</td><td>Leu</td><td>Asn</td>

<td>Ile 145</td><td>Pro</td><td>Gly</td><td>His</td><td>His</td><td>Pro 150</td><td>Ala</td><td>Arg</td><td>Ala</td><td>Met</td><td>His 155</td><td>Asp</td><td>Thr</td><td>Phe</td><td>Tyr</td><td>Phe 160</td>

<td>Asn</td><td>Ala</td><td>Asn</td><td>Met</td><td>Leu 165</td><td>Leu</td><td>Arg</td><td>Thr</td><td>His</td><td>Thr 170</td><td>Ser</td><td>Pro</td><td>Val</td><td>Gin</td><td>Val 175</td><td>Arg</td>

<td>Thr</td><td>Met</td><td>Glu</td><td>Ser 180</td><td>Gin</td><td>Gin</td><td>Pro</td><td>Pro</td><td>Ile 185</td><td>Arg</td><td>Ile</td><td>Val</td><td>Cys</td><td>Pro 190</td><td>Gly</td><td>Arg</td>

<td>Val</td><td>Tyr</td><td>Arg 195</td><td>Cys</td><td>Asp</td><td>Ser</td><td>Asp</td><td>Leu 200</td><td>Thr</td><td>His</td><td>Ser</td><td>Pro</td><td>Met 205</td><td>Phe</td><td>His</td><td>Gin</td>

<td>Val</td><td>Glu 210</td><td>Gly</td><td>Leu</td><td>Leu</td><td>Val</td><td>Asp 215</td><td>Glu</td><td>Gly</td><td>Val</td><td>Ser</td><td>Phe 220</td><td>Ala</td><td>Asp</td><td>Leu</td><td>Lys</td>

<td>Gly 225</td><td>Thr</td><td>Ile</td><td>Glu</td><td>Glu</td><td>Phe 230</td><td>Leu</td><td>Arg</td><td>Ala</td><td>Phe</td><td>Phe 235</td><td>Glu</td><td>Lys</td><td>Gin</td><td>Leu</td><td>Glu 240</td>

<td>Val</td><td>Arg</td><td>Phe</td><td>Arg</td><td>Pro 245</td><td>Ser</td><td>Phe</td><td>Phe</td><td>Pro</td><td>Phe 250</td><td>Thr</td><td>Glu</td><td>Pro</td><td>Ser</td><td>Ala 255</td><td>Glu</td>

<td>Val</td><td>Asp</td><td>Ile</td><td>Gin 260</td><td>Cys</td><td>Val</td><td>Ile</td><td>Cys</td><td>Ser 265</td><td>Gly</td><td>Asn</td><td>Gly</td><td>Cys</td><td>Arg 270</td><td>Val</td><td>Cys</td>

<td>Lys</td><td>Gin</td><td>Thr 275</td><td>Gly</td><td>Trp</td><td>Leu</td><td>Glu</td><td>Val 280</td><td>Met</td><td>Gly</td><td>Cys</td><td>Gly</td><td>Met 285</td><td>Val</td><td>His</td><td>Pro</td>

<td>Asn</td><td>Val 290</td><td>Leu</td><td>Arg</td><td>Met</td><td>Ser</td><td>Asn 295</td><td>Ile</td><td>Asp</td><td>Pro</td><td>Glu</td><td>Ly3 300</td><td>Phe</td><td>Gin</td><td>Gly</td><td>Phe</td>

<td>Ala 305</td><td>Phe</td><td>Gly</td><td>Met</td><td>Gly</td><td>Ala 310</td><td>Glu</td><td>Arg</td><td>Leu</td><td>Ala</td><td>Met 315</td><td>Leu</td><td>Arg</td><td>Tyr</td><td>Gly</td><td>Val 320</td>

<td>Asn Phe</td><td>Asp Arg</td><td>Leu</td><td>Arg</td><td>Leu 325</td><td>Phe</td><td>Phe</td><td>Asp</td><td>Asn</td><td>Asp 330</td><td>Leu</td><td>Arg</td><td>Phe</td><td>Leu</td><td>Gly 335</td><td>Gin</td>

&lt;210&gt; 82 &lt;211&gt; 785 &lt;212&gt; PRT &lt;213&gt; Thermus thermophilus &lt;400&gt; 82

-151-

<td>Met 1</td><td>Arg</td><td>Val</td><td>Pro</td><td>Phe 5</td><td>Ser</td><td>Trp</td><td>Leu</td><td>Lys</td><td>Ala 10</td><td>Tyr</td><td>Val</td><td>Pro</td><td>Glu</td><td>Leu 15</td><td>Glu</td>

<td>Ser</td><td>Pro</td><td>Glu</td><td>Val 20</td><td>Leu</td><td>Glu</td><td>Glu</td><td>Arg</td><td>Leu 25</td><td>Ala</td><td>Gly</td><td>Leu</td><td>Gly</td><td>Phe 30</td><td>Glu</td><td>Thr</td>

<td>Asp</td><td>Arg</td><td>ile 35</td><td>Glu</td><td>Arg</td><td>Val</td><td>Phe</td><td>Pro 40</td><td>Ile</td><td>Pro</td><td>Arg</td><td>Gly</td><td>Val 45</td><td>Val</td><td>Phe</td><td>Ala</td>

<td>Arg</td><td>Val 50</td><td>Leu</td><td>Glu</td><td>Ala</td><td>His</td><td>Pro 55</td><td>Ile</td><td>Pro</td><td>Gly</td><td>Thr</td><td>Arg 60</td><td>Leu</td><td>Lys</td><td>Arg</td><td>Leu</td>

<td>Val 65</td><td>Leu</td><td>Asp</td><td>Ala</td><td>Gly</td><td>Arg 70</td><td>Thr</td><td>Val</td><td>Glu</td><td>Val</td><td>Val 75</td><td>Ser</td><td>Gly</td><td>Ala</td><td>Glu</td><td>Asn 80</td>

<td>Ala</td><td>Arg</td><td>Lys</td><td>Gly</td><td>Ile 85</td><td>Gly</td><td>Val</td><td>Ala</td><td>Leu</td><td>Ala 90</td><td>Leu</td><td>Pro</td><td>Gly</td><td>Thr</td><td>Glu 95</td><td>Leu</td>

<td>Pro</td><td>Gly</td><td>Leu</td><td>Gly 100</td><td>Gin</td><td>Lys</td><td>val</td><td>Gly</td><td>Glu 105</td><td>Arg</td><td>Val</td><td>Ile</td><td>Gin</td><td>Gly 110</td><td>Val</td><td>Arg</td>

<td>Ser</td><td>Phe</td><td>Gly 115</td><td>Met</td><td>Ala</td><td>Leu</td><td>Ser</td><td>Pro 120</td><td>Arg</td><td>Glu</td><td>Leu</td><td>Gly</td><td>Val 125</td><td>Gly</td><td>Glu</td><td>Tyr</td>

<td>Gly</td><td>Gly 13 0</td><td>Gly</td><td>Leu</td><td>Leu</td><td>Glu</td><td>Phe 135</td><td>Pro</td><td>Glu</td><td>Asp</td><td>Ala</td><td>Leu 14 0</td><td>Pro</td><td>Pro</td><td>Gly</td><td>Thr</td>

<td>Pro 145</td><td>Leu</td><td>Ser</td><td>Glu</td><td>Ala</td><td>Trp 150</td><td>Pro</td><td>Glu</td><td>Glu</td><td>Val</td><td>val 155</td><td>Leu</td><td>Asp</td><td>Leu</td><td>Glu</td><td>Val 160</td>

<td>Thr</td><td>Pro</td><td>Asn</td><td>Arg</td><td>Pro</td><td>Asp</td><td>Ala</td><td>Leu</td><td>Gly</td><td>Leu</td><td>Leu</td><td>Gly</td><td>Leu</td><td>Ala</td><td>Arg</td><td>Asp</td>

-152-

<td>Leu</td><td>His</td><td>Ala</td><td>Leu</td><td>165 Gly</td><td>Tyr</td><td>Ala</td><td>Leu</td><td>Val</td><td>170 Glu</td><td>pro</td><td>GlU</td><td>Ala</td><td>Ala</td><td>175 Leu</td><td>Lys</td>

<td>Ala</td><td>Glu</td><td>Ala</td><td>180 Leu</td><td>Pro</td><td>Leu</td><td>Pro</td><td>Phe</td><td>185 Ala</td><td>Leu</td><td>Lya</td><td>Val</td><td>Glu</td><td>190 Asp</td><td>Pro</td><td>Glu</td>

<td>Gly</td><td>Ala</td><td>195 Pro</td><td>His</td><td>Phe</td><td>Thr</td><td>Leu</td><td>200 Gly</td><td>Tyr</td><td>Ala</td><td>Phe</td><td>Gly</td><td>205 Leu</td><td>Arg</td><td>Val</td><td>Ala</td>

<td>Pro</td><td>210 Ser</td><td>Pro</td><td>Leu</td><td>Trp</td><td>Met</td><td>215 Gin</td><td>Arg</td><td>Ala</td><td>Leu</td><td>Phe</td><td>220 Ala</td><td>Ala</td><td>Gly</td><td>Met</td><td>Arg</td>

<td>225 Pro</td><td>Ile</td><td>Asn</td><td>Asn</td><td>Val</td><td>230 Val</td><td>Asp</td><td>Val</td><td>Thr</td><td>Asn</td><td>235 Tyr</td><td>Val</td><td>Met</td><td>Leu</td><td>Glu</td><td>240 Arg</td>

<td>Ala</td><td>Gin</td><td>Pro</td><td>Met</td><td>245 His</td><td>Ala</td><td>Phe</td><td>Asp</td><td>Leu</td><td>250 Arg</td><td>Phe</td><td>Val</td><td>Gly</td><td>Glu</td><td>255 Gly</td><td>ile</td>

<td>Ala</td><td>Val</td><td>Arg</td><td>260 Arg</td><td>Ala</td><td>Arg</td><td>Glu</td><td>Gly</td><td>265 Glu</td><td>Arg</td><td>Leu</td><td>Lys</td><td>Thr</td><td>270 Leu</td><td>Asp</td><td>Gly</td>

<td>Val</td><td>Glu</td><td>275 Arg</td><td>Thr</td><td>Leu</td><td>His</td><td>Pro</td><td>280 Glu</td><td>Asp</td><td>Leu</td><td>Val</td><td>Ile</td><td>285 Ala</td><td>Gly</td><td>Trp</td><td>Arg</td>

<td>Gly</td><td>290 Glu</td><td>Glu</td><td>Ser</td><td>Phe</td><td>Pro</td><td>295 Leu</td><td>Gly</td><td>Leu</td><td>Ala</td><td>Gly</td><td>300 Val</td><td>Mec</td><td>Gly</td><td>Gly</td><td>Ala</td>

<td>305 Glu</td><td>Ser</td><td>Glu</td><td>Val</td><td>Arg</td><td>310 Glu</td><td>Asp</td><td>Thr</td><td>Glu</td><td>Ala</td><td>315 Ile</td><td>Ala</td><td>Leu</td><td>Glu</td><td>Val</td><td>320 Ala</td>

<td>Cys</td><td>Phe</td><td>Asp</td><td>Pro</td><td>325 Val</td><td>Ser</td><td>Ile</td><td>Arg</td><td>Lys</td><td>330 Thr</td><td>Ala</td><td>Arg</td><td>Arg</td><td>His</td><td>335 Gly</td><td>Leu</td>

<td>Arg</td><td>Thr</td><td>Glu</td><td>340 Ala</td><td>Ser</td><td>His</td><td>Arg</td><td>Phe</td><td>345 GlU</td><td>Arg</td><td>Gly</td><td>Val</td><td>Asp</td><td>350 pro</td><td>Leu</td><td>Gly</td>

<td>Gin</td><td>Val</td><td>355 Pro</td><td>Ala</td><td>Gin</td><td>Arg</td><td>Arg</td><td>360 Ala</td><td>Leu</td><td>Ser</td><td>Leu</td><td>Leu</td><td>365 Gin</td><td>Ala</td><td>Leu</td><td>Ala</td>

<td>Gly</td><td>370 Ala</td><td>Arg</td><td>Val</td><td>Ala</td><td>Glu</td><td>375 Ala</td><td>Leu</td><td>Leu</td><td>Glu</td><td>Ala</td><td>380 Gly</td><td>Ser</td><td>Pro</td><td>Lys</td><td>Pro</td>

<td>385 Pro</td><td>Glu</td><td>Ala</td><td>Ile</td><td>Pro</td><td>390 Phe</td><td>Arg</td><td>Pro</td><td>Glu</td><td>Tyr</td><td>395 Ala</td><td>Asn</td><td>Arg</td><td>Leu</td><td>Leu</td><td>400 Gly</td>

<td>Thr</td><td>Ser</td><td>Tyr</td><td>Pro</td><td>405 Glu</td><td>Ala</td><td>Glu</td><td>Gin</td><td>Ile</td><td>410 Ala</td><td>Ile</td><td>Leu</td><td>Lye</td><td>Arg</td><td>415 Leu</td><td>Gly</td>

<td>Cys</td><td>Arg</td><td>Val</td><td>420 Glu</td><td>Gly</td><td>Glu</td><td>Gly</td><td>Pro</td><td>425 Thr</td><td>Tyr</td><td>Arg</td><td>Val</td><td>Thr</td><td>430 Pro</td><td>Pro</td><td>Ser</td>

<td>His</td><td>Arg</td><td>435 Leu</td><td>Asp</td><td>Leu</td><td>Arg</td><td>Leu</td><td>440 Glu</td><td>Glu</td><td>Asp</td><td>Leu</td><td>Val</td><td>445 Glu</td><td>Glu</td><td>val</td><td>Ala</td>

<td>Arg</td><td>450 Ile</td><td>Gin</td><td>Gly</td><td>Tyr</td><td>Glu</td><td>455 Thr</td><td>Ile</td><td>Pro</td><td>Leu</td><td>Ala</td><td>460 Leu</td><td>Pro</td><td>Ala</td><td>Phe</td><td>Phe</td>

<td>465 Pro</td><td>Ala.</td><td>Pro</td><td>Asp</td><td>Asn</td><td>470 Arg</td><td>Gly</td><td>Val</td><td>Glu</td><td>Ala</td><td>475 Pro</td><td>Tyr</td><td>Arg</td><td>Lys</td><td>Glu</td><td>480 Gin</td>

<td>Arg</td><td>Leu</td><td>Arg</td><td>Glu</td><td>485 Val</td><td>Leu</td><td>Ser</td><td>Gly</td><td>Leu</td><td>490 Gly</td><td>Phe</td><td>Gin</td><td>Glu</td><td>Val</td><td>495 Tyr</td><td>Thr</td>

<td>Tyr</td><td>Ser</td><td>Phe</td><td>500 Met</td><td>Asp</td><td>Pro</td><td>Glu</td><td>Asp</td><td>505 Ala</td><td>Arg</td><td>Arg</td><td>Phe</td><td>Arg</td><td>510 Leu</td><td>Asp</td><td>Pro</td>

<td>Pro</td><td>Arg</td><td>515 Leu</td><td>Leu</td><td>Leu</td><td>Leu</td><td>Asn</td><td>520 Pro</td><td>Leu</td><td>Ala</td><td>Pro</td><td>Glu</td><td>525 Lys</td><td>Ala</td><td>Ala</td><td>Leu</td>

<td>Arg</td><td>530 Thr</td><td>His</td><td>Leu</td><td>Phe</td><td>Pro</td><td>535 Gly</td><td>Leu</td><td>Val</td><td>Arg</td><td>Val</td><td>540 Leu</td><td>Lys</td><td>Glu</td><td>Asn</td><td>Leu</td>

<td>545 Asp</td><td>Leu</td><td>Asp</td><td>Arg</td><td>Pro</td><td>550 Glu</td><td>Arg</td><td>Ala</td><td>Leu</td><td>Leu</td><td>555 Phe</td><td>Glu</td><td>Val</td><td>Gly</td><td>Arg</td><td>560 Val</td>

<td>Phe</td><td>Arg</td><td>Glu</td><td>Arg</td><td>565 Glu</td><td>Glu</td><td>Thr</td><td>His</td><td>Leu</td><td>570 Ala</td><td>Gly</td><td>Leu</td><td>Leu</td><td>Phe</td><td>575 Gly</td><td>Glu</td>

<td>Gly</td><td>Val</td><td>Gly</td><td>580 Leu</td><td>Pro</td><td>Trp</td><td>Ala</td><td>Lys</td><td>585 GlU</td><td>Arg</td><td>Leu</td><td>Ser</td><td>Gly</td><td>590 Tyr</td><td>Phe</td><td>Leu</td>

<td>Leu</td><td>Lys</td><td>595 Gly</td><td>Tyr</td><td>Leu</td><td>Glu</td><td>Ala</td><td>600 Leu</td><td>Phe</td><td>Ala</td><td>Arg</td><td>Leu</td><td>605 Gly</td><td>Leu</td><td>Ala</td><td>Phe</td>

<td>Arg</td><td>SIO val</td><td>Glu</td><td>Ala</td><td>Gin</td><td>Ala</td><td>615 Phe</td><td>Pro</td><td>Phe</td><td>Leu</td><td>His</td><td>620 Pro</td><td>Gly</td><td>Val</td><td>Ser</td><td>Gly</td>

-153625 630 535 640

<td>Arg</td><td>Val</td><td>Leu</td><td>Val</td><td>Glu 645</td><td>Gly</td><td>Glu</td><td>Glu</td><td>Val</td><td>Gly 650</td><td>Phe</td><td>Leu</td><td>Gly</td><td>Ala</td><td>Leu 655</td><td>His</td>

<td>Pro</td><td>Glu</td><td>Ile</td><td>Ala 560</td><td>Gin</td><td>Glu</td><td>Leu</td><td>Glu</td><td>Leu 665</td><td>Pro</td><td>Pro</td><td>Val</td><td>His</td><td>Leu 670</td><td>Phe</td><td>Glu</td>

<td>Leu</td><td>Arg</td><td>Leu 675</td><td>Pro</td><td>Leu</td><td>Pro</td><td>Asp</td><td>Lys 680</td><td>Pro</td><td>Leu</td><td>Ala</td><td>Phe</td><td>Gin 685</td><td>Asp</td><td>pro</td><td>Ser</td>

<td>Arg</td><td>His €90</td><td>Pro</td><td>Ala</td><td>Ala</td><td>Phe</td><td>Arg 6 95</td><td>Asp</td><td>Leu</td><td>Ala</td><td>Val</td><td>Val 700</td><td>Val</td><td>Pro</td><td>Ala</td><td>Pro</td>

<td>Thr 705</td><td>pro</td><td>Tyr</td><td>Gly</td><td>Glu</td><td>Val 710</td><td>Glu</td><td>Ala</td><td>Leu</td><td>Val</td><td>Arg 715</td><td>Glu</td><td>Ala</td><td>Ala</td><td>Gly</td><td>Pro 720</td>

<td>Tyr</td><td>Leu</td><td>Glu</td><td>Ser</td><td>Leu 725</td><td>Ala</td><td>Leu</td><td>Phe</td><td>Asp</td><td>Leu 730</td><td>Tyr</td><td>Gin</td><td>Gly</td><td>Pro</td><td>Pro 735</td><td>Leu</td>

<td>Pro</td><td>Glu</td><td>Gly</td><td>Hi 3 740</td><td>Lys</td><td>Ser</td><td>Leu</td><td>Ala</td><td>Phe 745</td><td>His</td><td>Leu</td><td>Arg</td><td>Phe</td><td>Arg 7,5 0</td><td>His</td><td>Pro</td>

<td>Lye</td><td>Arg</td><td>Thr 755</td><td>Leu</td><td>Arg</td><td>Asp</td><td>Glu</td><td>Glu 760</td><td>Val</td><td>Glu</td><td>Glu</td><td>Ala</td><td>Val 765</td><td>Ser</td><td>Arg</td><td>Val</td>

<td>Ala</td><td>Glu</td><td>Ala</td><td>Leu</td><td>Arg</td><td>Ala-</td><td>Arg</td><td>Gly</td><td>Phe</td><td>Gly</td><td>Leu</td><td>Arg</td><td>Gly</td><td>Leu</td><td>Asp</td><td>Thr</td>

770 775 780

Pro

785 &lt;210&gt; 83 &lt;211&gt; 598 &lt;212&gt; PRT &lt;213&gt; Arabidopsis thaliana &lt;400&gt; 83

<td>Met 1</td><td>Pro</td><td>Thr</td><td>Ile</td><td>Ser 5</td><td>Val</td><td>Gly</td><td>Arg</td><td>Asp</td><td>Arg 10</td><td>Leu</td><td>Phe</td><td>Ala</td><td>Ala</td><td>Leu 15</td><td>Gly</td>

<td>Glu</td><td>Ser</td><td>Tyr</td><td>Thr 20</td><td>Gin</td><td>Glu</td><td>Lys</td><td>Phe</td><td>Glu 25</td><td>Glu</td><td>Leu</td><td>Cys</td><td>Phe</td><td>Ser 30</td><td>Phe</td><td>Gly</td>

<td>Ile</td><td>Glu</td><td>Leu 35</td><td>Asp</td><td>Asp</td><td>Val</td><td>Thr</td><td>Thr 40</td><td>Glu</td><td>Lys</td><td>Ala</td><td>Ile</td><td>Ile 45</td><td>Arg</td><td>Lys</td><td>Glu</td>

<td>Lys</td><td>His 50</td><td>Ile</td><td>Asp</td><td>Glu</td><td>Glu</td><td>Ala 55</td><td>Asp</td><td>ASp</td><td>Asp</td><td>Glu</td><td>Glu 60</td><td>Ile</td><td>Ile</td><td>Tyr</td><td>Lys</td>

<td>Ile 65</td><td>Glu</td><td>Ile</td><td>Pro</td><td>Ala</td><td>Asn 70</td><td>Arg</td><td>Pro</td><td>Asp</td><td>Leu</td><td>Leu 75</td><td>Cys</td><td>Leu</td><td>Glu</td><td>Gly</td><td>Leu 80</td>

<td>Ala</td><td>Gin</td><td>Ser</td><td>Leu</td><td>Arg 85</td><td>Val</td><td>Phe</td><td>Ile</td><td>Glu</td><td>Lys 90</td><td>Gin</td><td>Glu</td><td>Ile</td><td>Pro</td><td>Thr 95</td><td>Tyr</td>

<td>Thr</td><td>Leu</td><td>Ala</td><td>Asp 100</td><td>Ile</td><td>Ser</td><td>Lys</td><td>Asp</td><td>Lys 105</td><td>Ile</td><td>Leu</td><td>Gin</td><td>Met</td><td>Asn 110</td><td>Val</td><td>Lys</td>

<td>Pro</td><td>Glu</td><td>Thr 115</td><td>Ser</td><td>Lys</td><td>Ile</td><td>Arg</td><td>Pro 120</td><td>Phe</td><td>Val</td><td>Val</td><td>Cys</td><td>Ala 125</td><td>Val</td><td>Leu</td><td>Arg</td>

<td>Gly</td><td>Val 130</td><td>Thr</td><td>Phe</td><td>Asp</td><td>Glu</td><td>Ala 135</td><td>Arg</td><td>Tyr</td><td>Asn</td><td>Ser</td><td>Phe 14 0</td><td>Ile</td><td>Asp</td><td>Leu</td><td>Gin</td>

<td>Asp 145</td><td>Lys</td><td>Leu</td><td>His</td><td>Gin</td><td>Asn 150</td><td>Ile</td><td>Cys</td><td>Arg</td><td>Arg</td><td>Arg 155</td><td>Ser</td><td>Leu</td><td>Val</td><td>Ala</td><td>Ile 160</td>

<td>Gly</td><td>Thr</td><td>His</td><td>Asp</td><td>Leu 165</td><td>Asp</td><td>Thr</td><td>Leu</td><td>Gin</td><td>Gly 170</td><td>Pro</td><td>Phe</td><td>Thr</td><td>Tyr</td><td>Glu 175</td><td>Ala</td>

<td>Leu</td><td>Pro</td><td>Pro</td><td>Thr 180</td><td>Asp</td><td>Ile</td><td>Asn</td><td>Phe</td><td>Val 185</td><td>Pro</td><td>Leu</td><td>Lys</td><td>Gin</td><td>Thr 190</td><td>Lys</td><td>Ser</td>

<td>Phe</td><td>Arg</td><td>Ala 195</td><td>Asp</td><td>Glu</td><td>Leu</td><td>Ile</td><td>Glu 200</td><td>Phe</td><td>Tyr</td><td>Lys</td><td>Ser</td><td>Asp 205</td><td>Met</td><td>Lys</td><td>Leu</td>

<td>Lys</td><td>Lys 210</td><td>Phe</td><td>Leu</td><td>His</td><td>Ile</td><td>Ile 215</td><td>Glu</td><td>Asn</td><td>Ser</td><td>Pro</td><td>Val 220</td><td>Phe</td><td>Pro</td><td>Val</td><td>Leu</td>

<td>Tyr</td><td>Asp</td><td>Ser</td><td>Lys</td><td>Arg</td><td>Thr</td><td>Val</td><td>Leu</td><td>Ser</td><td>Leu</td><td>Pro</td><td>Pro</td><td>Ile</td><td>Ile</td><td>Asn</td><td>Gly</td>

-154•«ο

<td>225 Ala</td><td>His</td><td colspan="2">Ser Ala</td><td>Ile 24 5</td><td colspan="4">230 Thr Leu Gln Thr</td><td>Lys 250</td><td>235 Asn</td><td>val</td><td>Phe</td><td>ile</td><td>Glu 255</td><td>240 Cys</td>

<td>Thr</td><td>Ala</td><td>Thr</td><td>Asp</td><td>Leu</td><td>Thr</td><td>Lys</td><td>Ala</td><td>Lys</td><td>Ile</td><td>Val</td><td>Leu</td><td>Asn</td><td>Thr</td><td>Met</td><td>Val</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>Thr</td><td>Thr</td><td>Phe</td><td>Ser</td><td>Glu</td><td>Phe</td><td>Cys</td><td>Ala</td><td>Arg</td><td>Lys</td><td>Phe</td><td>Glu</td><td>Ile</td><td>Glu</td><td>Pro</td><td>Val</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>Glu</td><td>Val</td><td>Thr</td><td>Tyr</td><td>Asp</td><td>Asp</td><td>Gly</td><td>Lys</td><td>Ser</td><td>Tyr</td><td>Ile</td><td>Tyr</td><td>Pro</td><td>Asp</td><td>Leu</td><td>Ala</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>Val</td><td>Tyr</td><td>Asp</td><td>Met</td><td>Glu</td><td>val</td><td>Pro</td><td>Leu</td><td>Ser</td><td>Phe</td><td>Ile</td><td>Thr</td><td>Asp</td><td>Ser</td><td>Ile</td><td>Gly</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

<td>Val</td><td>Ser</td><td>Leu</td><td>Lys</td><td>Val</td><td>Glu</td><td>Gln</td><td>Val</td><td>Thr</td><td>Ser</td><td>Leu</td><td>Leu</td><td>Thr</td><td>Arg</td><td>Met</td><td>Gln</td>

<td></td><td></td><td></td><td></td><td>32S</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td>

<td>Leu</td><td>Gln</td><td>Ala</td><td>Glu</td><td>Gln</td><td>Ala</td><td>Lys</td><td>Ser</td><td>Ser</td><td>Asp</td><td>Asn</td><td>Gln</td><td>Cys</td><td>Ala</td><td>Ile</td><td>Lys</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td>

<td>Val</td><td>His</td><td>Val</td><td>pro</td><td>Pro</td><td>Ser</td><td>Arg</td><td>Ser</td><td>ASp</td><td>val</td><td>Leu</td><td>His</td><td>Pro</td><td>cys</td><td>Asp</td><td>Val</td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td>

<td>Met</td><td>Glu</td><td>Asp</td><td>Val</td><td>Ala</td><td>Ile</td><td>Ala</td><td>Tyr</td><td>Gly</td><td>Phe</td><td>Asn</td><td>Asn</td><td>Ile</td><td>Pro</td><td>Thr</td><td>Arg</td>

<td></td><td> 370</td><td></td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td>

<td>Lys</td><td>Pro</td><td>Ala</td><td>Ser</td><td>Ile</td><td>Lys</td><td>Pro</td><td>Leu</td><td>Thr</td><td>Leu</td><td>Asn</td><td>Glu</td><td>Leu</td><td>Thr</td><td>Asp</td><td>Leu</td>

<td> 335</td><td></td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td> 3 95</td><td></td><td></td><td></td><td></td><td> 400</td>

<td>Leu</td><td>Arg</td><td>Ile</td><td>Glu</td><td>Ile</td><td>Ala</td><td>Met</td><td>Cys</td><td>val</td><td>Tyr</td><td>Thr</td><td>Glu</td><td>Val</td><td>val</td><td>Thr</td><td>Trp</td>

<td></td><td></td><td></td><td></td><td> 405</td><td></td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td>

<td>Leu</td><td>Leu</td><td>Cys</td><td>Ser</td><td>His</td><td>Lys</td><td>Glu</td><td>Asn</td><td>Phe</td><td>Ala</td><td>Met</td><td>Leu</td><td>Asn</td><td>Arg</td><td>Glu</td><td>Asp</td>

<td></td><td></td><td></td><td> 420</td><td></td><td></td><td></td><td></td><td> 425</td><td></td><td></td><td></td><td></td><td> 430</td><td></td><td></td>

<td>Val</td><td>Asn</td><td>Ser</td><td>Ala</td><td>Val</td><td>ile</td><td>Val</td><td>Gly</td><td>Asn</td><td>Pro</td><td>Arg</td><td>Ser</td><td>Ala</td><td>Asp</td><td>Phe</td><td>Glu</td>

<td></td><td></td><td> 435</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td></td><td></td><td></td>

<td>Ala</td><td>Met</td><td>Arg</td><td>Arg</td><td>Ala</td><td>Leu</td><td>Met</td><td>Pro.</td><td>Gly</td><td>Leu</td><td>Leu</td><td>Lys</td><td>Thr</td><td>Val</td><td>Gly</td><td>His</td>

<td></td><td> 450</td><td></td><td></td><td></td><td></td><td> 455</td><td></td><td></td><td></td><td></td><td> 460</td><td></td><td></td><td></td><td></td>

<td>Asn</td><td>Asn</td><td>Lys</td><td>Tyr</td><td>Pro</td><td>Lys</td><td>Pro</td><td>Ile</td><td>Lys</td><td>Ile</td><td>Phe</td><td>Glu</td><td>Ile</td><td>Ser</td><td>Asp</td><td>val</td>

<td> 465</td><td></td><td></td><td></td><td></td><td> 470</td><td></td><td></td><td></td><td></td><td> 475</td><td></td><td></td><td></td><td></td><td> 480</td>

<td>Val</td><td>Met</td><td>Leu</td><td>Asp</td><td>Glu</td><td>Ser</td><td>Lys</td><td>Asp</td><td>Val</td><td>Gly</td><td>Ala</td><td>Ser</td><td>Asn</td><td>Arg</td><td>Arg</td><td>His</td>

<td></td><td></td><td></td><td></td><td> 485</td><td></td><td></td><td></td><td></td><td> 490</td><td></td><td></td><td></td><td></td><td> 495</td><td></td>

<td>Leu</td><td>Ala</td><td>Ala</td><td>Leu</td><td>Tyr</td><td>Cys</td><td>Gly</td><td>Ala</td><td>Thr</td><td>Ser</td><td>Gly</td><td>Phe</td><td>Glu</td><td>Leu</td><td>Ile</td><td>His</td>

<td></td><td></td><td></td><td> 500</td><td></td><td></td><td></td><td></td><td> 505</td><td></td><td></td><td></td><td></td><td> 510</td><td></td><td></td>

<td>Gly</td><td>Leu</td><td>Val</td><td>Asp</td><td>Arg</td><td>Ile</td><td>Met</td><td>Glu</td><td>vai</td><td>Met</td><td>Ala</td><td>Ile</td><td>Pro</td><td>Phe</td><td>Leu</td><td>Thr</td>

<td></td><td></td><td> 515</td><td></td><td></td><td></td><td></td><td> 520</td><td></td><td></td><td></td><td></td><td> 525</td><td></td><td></td><td></td>

<td>Ile</td><td>His</td><td>Glu</td><td>Asn</td><td>Asn</td><td>Val</td><td>Pro</td><td>Ile</td><td>Asn</td><td>Glu</td><td>Lys</td><td>Asp</td><td>Gly</td><td>Tyr</td><td>Tyr</td><td>val</td>

<td></td><td> 530</td><td></td><td></td><td></td><td></td><td> 53 5</td><td></td><td></td><td></td><td></td><td> 540</td><td></td><td></td><td></td><td></td>

<td>Lys</td><td>Leu</td><td>Ser</td><td>Gln</td><td>Glu</td><td>Pro</td><td>Glu</td><td>Phe</td><td>Leu</td><td>Pro</td><td>Gly</td><td>Arg</td><td>Gln</td><td>Ala</td><td>Ser</td><td>Ile</td>

<td> 545</td><td></td><td></td><td></td><td></td><td> 550</td><td></td><td></td><td></td><td></td><td> 555</td><td></td><td></td><td></td><td></td><td> 560</td>

<td>Ile</td><td>Val</td><td>Arg</td><td>Gly</td><td>Lys</td><td>His</td><td>Ile</td><td>Gly</td><td>Asn</td><td>Phe</td><td>Gly</td><td>Ile</td><td>Val</td><td>His</td><td>Pro</td><td>Glu</td>

<td></td><td></td><td></td><td></td><td>5S5</td><td></td><td></td><td></td><td></td><td> 570</td><td></td><td></td><td></td><td></td><td> 575</td><td></td>

<td>Val</td><td>Leu</td><td>Asn</td><td>Asn</td><td>Phe</td><td>Asp</td><td>Ile</td><td>Pro</td><td>Asp</td><td>Pro</td><td>Cys</td><td>Ser</td><td>Tyr</td><td>Leu</td><td>Glu</td><td>Leu</td>

<td></td><td></td><td></td><td> 580</td><td></td><td></td><td></td><td></td><td> 585</td><td></td><td></td><td></td><td></td><td> 590</td><td></td><td></td>

<td>Asp</td><td>Ile</td><td>Glu</td><td>Ala</td><td>Ile</td><td>Leu</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td> 595</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 84 &lt;211&gt;589 &lt;212&gt; PRT &lt;213&gt; Drosophila melanogaster &lt;400&gt; 84

Met Pro Thr Ile Gly Val Lya Arg Asp Leu Leu Phe Glu Ala Leu Gly

10 15

Lys Thr Tyr Thr Aap Asp Glu Phe Gln Asp Leu Cys Phe Ala Phe Gly

-155-

<td>Leu</td><td>Glu</td><td>Leu</td><td>20 Asp</td><td>Glu</td><td>Val</td><td>Thr</td><td>Thr</td><td>25 Glu</td><td>Lys</td><td>Gin</td><td>Met</td><td>Leu</td><td>30 Thr</td><td>Lys</td><td>Glu</td>

<td>Gin</td><td>Gly</td><td>35 ASP</td><td>Val</td><td>Ala</td><td>Ala</td><td>Ala</td><td>40 Ala</td><td>Asn</td><td>Ala</td><td>Ser</td><td>Glu</td><td>45 Glu</td><td>Ile</td><td>ile</td><td>Tyr</td>

<td>Arg</td><td>50 Ile</td><td>Asp</td><td>Ile</td><td>Pro</td><td>Ala</td><td>55 Asn</td><td>Arg</td><td>Tyr</td><td>Asp</td><td>Leu</td><td>60 Leu</td><td>Cys</td><td>Leu</td><td>Glu</td><td>Gly</td>

<td>65 Leu</td><td>Val</td><td>Thr</td><td>Gly</td><td>Leu</td><td>70 Leu</td><td>val</td><td>Phe</td><td>Gin</td><td>Gly</td><td>75 Lys</td><td>Leu</td><td>Lys</td><td>Pro</td><td>Pro</td><td>80 Lys</td>

<td>Phe</td><td>Gin</td><td>Phe</td><td>Val</td><td>85 Glu</td><td>Leu</td><td>Ala</td><td>Lys</td><td>Arg</td><td>90 Gin</td><td>Val</td><td>Leu</td><td>Lys</td><td>Ile</td><td>95 Asp</td><td>Pro</td>

<td>Ser</td><td>Thr</td><td>Ala</td><td>100 Gin</td><td>rie</td><td>Arg</td><td>Pro</td><td>Tyr</td><td>105 Ala</td><td>Val</td><td>Ala</td><td>Ala</td><td>Val</td><td>110 Leu</td><td>Arg</td><td>Asn</td>

<td>Val</td><td>Thr</td><td>115 Phe</td><td>Thr</td><td>Gin</td><td>Ala</td><td>Ser</td><td>120 Tyr</td><td>Asn</td><td>Ser</td><td>Phe</td><td>Ile</td><td>125 Asp</td><td>Leu</td><td>Gin</td><td>Asp</td>

<td>Lys</td><td>130 Leu</td><td>His</td><td>Gin</td><td>Asn</td><td>Ile</td><td>135 Cys</td><td>Arg</td><td>Lys</td><td>Arg</td><td>Thr</td><td>140 Leu</td><td>Val</td><td>Ala</td><td>Ile</td><td>Gly</td>

<td>145 Thr</td><td>His</td><td>Asp</td><td>Leu</td><td>Asp</td><td>ISO Thr</td><td>Leu</td><td>Gin</td><td>Gly</td><td>Pro</td><td>155 Phe</td><td>Ser</td><td>Tyr</td><td>Glu</td><td>Ala</td><td>160 Leu</td>

<td>Ala</td><td>Pro</td><td>Asp</td><td>Gin</td><td>165 Ile</td><td>Lys</td><td>Phe</td><td>Lys</td><td>Pro</td><td>170 Leu</td><td>Asn</td><td>Gin</td><td>Thr</td><td>Lys</td><td>175 Glu</td><td>Met</td>

<td>Thr</td><td>Gly</td><td>Ser</td><td>180 Glu</td><td>Leu</td><td>Met</td><td>Asp</td><td>Phe</td><td>185 Tyr</td><td>Ser</td><td>Thr</td><td>His</td><td>Ala</td><td>190 Gin</td><td>Leu</td><td>Lys</td>

<td>Gin</td><td>Tyr</td><td>195 Leu</td><td>Pro</td><td>Ile</td><td>ile</td><td>Arg</td><td>200 Glu</td><td>Ser</td><td>Pro</td><td>Val</td><td>Tyr</td><td>205 Pro</td><td>Val</td><td>Ile</td><td>Tyr.</td>

<td>Asp</td><td>210 Ala</td><td>Asn</td><td>Arg</td><td>Val</td><td>Val</td><td>215 Leu</td><td>Ser</td><td>Leu</td><td>Pro</td><td>Pro</td><td>220 Ile</td><td>Ile</td><td>Asn</td><td>Gly</td><td>Asp</td>

<td>225 His</td><td>Ser</td><td>Lys</td><td>Ile</td><td>Thr</td><td>230 Leu</td><td>Lys</td><td>Thr</td><td>Lys</td><td>Asn</td><td>235 Val</td><td>Phe</td><td>Ile</td><td>Glu</td><td>Cys</td><td>240 Thr</td>

<td>Ala</td><td>Thr</td><td>Asp</td><td>Arg</td><td>245 Thr</td><td>Lys</td><td>Ala</td><td>Lys</td><td>Val</td><td>250 Val</td><td>Leu</td><td>Asp</td><td>Thr</td><td>Ile</td><td>255 Val</td><td>Cys</td>

<td>Leu</td><td>Phe</td><td>Ser</td><td>260 Glu</td><td>His</td><td>Cys</td><td>Ala</td><td>Gin</td><td>265 Lys</td><td>Phe</td><td>Thr</td><td>Val</td><td>Glu</td><td>270 Pro</td><td>Cys</td><td>Asp</td>

<td>Val</td><td>Val</td><td>275 Gin</td><td>Pro</td><td>Asp</td><td>Gly</td><td>Ser</td><td>280 val</td><td>Ile</td><td>Ser</td><td>Tyr</td><td>Pro</td><td>285 Glu</td><td>Leu</td><td>Glu</td><td>Val</td>

<td>Arg</td><td>290 Glu</td><td>Glu</td><td>Arg</td><td>Ile</td><td>Ser</td><td>295 Val</td><td>Lys</td><td>Arg</td><td>Ala</td><td>Asn</td><td>300 Ala</td><td>Tyr</td><td>Ile</td><td>Gly</td><td>Ile</td>

<td>305 Asp</td><td>Glu</td><td>Pro</td><td>Ala</td><td>Glu</td><td>310 Lys</td><td>Leu</td><td>Ala</td><td>Asp</td><td>Met</td><td>315 Leu</td><td>Thr</td><td>Arg</td><td>Met</td><td>Tyr</td><td>320 Leu</td>

<td>Glu</td><td>Ala</td><td>Lys</td><td>Val</td><td>325 Asp</td><td>Gly</td><td>Asp</td><td>Ser</td><td>Leu</td><td>330 Val</td><td>Val</td><td>Lys</td><td>Ile</td><td>Pro</td><td>335 Pro</td><td>Thr</td>

<td>Arg</td><td>His</td><td>Asp</td><td>340 Val</td><td>Ile</td><td>His</td><td>Ala</td><td>Cys</td><td>345 ASp</td><td>Ile</td><td>Tyr</td><td>Glu</td><td>Asp</td><td>350 Val</td><td>Ala</td><td>Ile</td>

<td>Ala</td><td>Tyr</td><td>3S5 Gly</td><td>Tyr</td><td>Asn</td><td>Asn</td><td>Ile</td><td>360 Lys</td><td>Lys</td><td>Ser</td><td>Leu</td><td>Pro</td><td>365 Ala</td><td>Phe</td><td>Met</td><td>Gin</td>

<td>Ile</td><td>370 Ala</td><td>Lys</td><td>Gin</td><td>Phe</td><td>Pro</td><td>375 Leu</td><td>Asn</td><td>Lys</td><td>Leu</td><td>Thr</td><td>380 Glu</td><td>Gin</td><td>Leu</td><td>Arg</td><td>Glu</td>

<td>385 Gin</td><td>Val</td><td>Ala</td><td>Gin</td><td>Ala</td><td>390 Gly</td><td>Phe</td><td>Thr</td><td>Glu</td><td>Ala</td><td>395 Leu</td><td>Thr</td><td>Phe</td><td>Thr</td><td>Leu</td><td>400 Cys</td>

<td>Ser</td><td>Arg</td><td>ASp</td><td>Asp</td><td>405 ile</td><td>Gly</td><td>Arg</td><td>Lys</td><td>Leu</td><td>410 Asn</td><td>Lys</td><td>Asn</td><td>Ile</td><td>Asp</td><td>415 Ala</td><td>Leu</td>

<td>Pro</td><td>Ala</td><td>val</td><td>420 HiS</td><td>Ile</td><td>Gly</td><td>Asn</td><td>Pro</td><td>425 Lys</td><td>Thr</td><td>Leu</td><td>Glu</td><td>Phe</td><td>430 Gin</td><td>Val</td><td>Val</td>

<td>Arg</td><td>Thr</td><td>435 Thr</td><td>Leu</td><td>Leu</td><td>Pro</td><td>Gly</td><td>440 Leu</td><td>Leu</td><td>Lys</td><td>Thr</td><td>Leu</td><td>445 val</td><td>Ala</td><td>Asn</td><td>Arg</td>

<td>Lya</td><td>450 Het</td><td>Pro</td><td>Leu</td><td>Pro</td><td>Leu</td><td>455 Lys</td><td>Leu</td><td>Phe</td><td>Glu</td><td>Ile</td><td>460 Ser</td><td>Asp</td><td>val</td><td>Val</td><td>Val</td>

<td>465 Ala</td><td>ASp</td><td>Glu</td><td>Ser</td><td>Thr</td><td>470 GlU</td><td>Val</td><td>Gly</td><td>Ala</td><td>Arg</td><td>475 Asn</td><td>Glu</td><td>Arg</td><td>Arg</td><td>val</td><td>480 Cys</td>

<td></td><td></td><td></td><td></td><td> 485</td><td></td><td></td><td></td><td></td><td> 490</td><td></td><td></td><td></td><td></td><td> 495</td><td></td>

<td>Ala</td><td>Val</td><td>Asn</td><td>Cys</td><td>Asn</td><td>Lys</td><td>Thr</td><td>Ala</td><td>Gly</td><td>Phe</td><td>Glu</td><td>Val</td><td>Val</td><td>His</td><td>Gly</td><td>Leu</td>

<td></td><td></td><td></td><td> 500</td><td></td><td></td><td></td><td></td><td> 505</td><td></td><td></td><td></td><td></td><td> 510</td><td></td><td></td>

<td>Leu</td><td>Asp</td><td>Arg</td><td>Val</td><td>Met</td><td>Gin</td><td>Leu</td><td>Leu</td><td>Ser</td><td>Val</td><td>Pro</td><td>Trp</td><td>Lys</td><td>Ser</td><td>Ala</td><td>Ser</td>

<td></td><td></td><td> 515</td><td></td><td></td><td></td><td></td><td> 520</td><td></td><td></td><td></td><td></td><td> 525</td><td></td><td></td><td></td>

<td>Gly</td><td>Thr</td><td>Lys</td><td>Gly</td><td>Tyr</td><td>Tyr</td><td>Leu</td><td>Gin</td><td>Ala</td><td>Thr</td><td>Glu</td><td>Asp</td><td>Pro</td><td>ser</td><td>Tyr</td><td>Phe</td>

<td></td><td> 530</td><td></td><td></td><td></td><td></td><td> 535</td><td></td><td></td><td></td><td></td><td> 540</td><td></td><td></td><td></td><td></td>

<td>Pro</td><td>Gly</td><td>Arg</td><td>Cys</td><td>Ala</td><td>Asn</td><td>Val</td><td>Met</td><td>Tyr</td><td>ASp</td><td>Gly</td><td>Val</td><td>Val</td><td>Ile</td><td>Gly</td><td>Lys</td>

<td> 545</td><td></td><td></td><td></td><td></td><td> 550</td><td></td><td></td><td></td><td></td><td> 555</td><td></td><td></td><td></td><td></td><td> 560</td>

<td>Ile</td><td>Gly</td><td>Val</td><td>Leu</td><td>Hi3</td><td>Pro</td><td>Thr</td><td>Val</td><td>Leu</td><td>Gin</td><td>Ala</td><td>Phe</td><td>Glu</td><td>Leu</td><td>Thr</td><td>Thr</td>

<td></td><td></td><td></td><td></td><td> 565</td><td></td><td></td><td></td><td></td><td> 570</td><td></td><td></td><td></td><td></td><td> 575</td><td></td>

<td>Pro</td><td>Cys</td><td>Ser</td><td>Ala</td><td>Val</td><td>Glu</td><td>Phe</td><td>Thr</td><td>Ile</td><td>Glu</td><td>Pro</td><td>Phe</td><td>Val</td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> 580</td><td></td><td></td><td></td><td></td><td> 585</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 85

-156&lt;211&gt; 591 &lt;212&gt; PRT &lt;213&gt; Caenorhabditis elegans &lt;400&gt; 85

<td>Met 1</td><td>Pro</td><td>Thr</td><td>Val</td><td>Gly 5</td><td>ile</td><td>Lys</td><td>Lys</td><td>Val</td><td>Ile 10</td><td>Leu</td><td>Asp</td><td>Lys</td><td>His</td><td>Phe 15</td><td>Lys</td>

<td>Arg</td><td>Val</td><td>Tyr</td><td>Ser 20</td><td>Glu</td><td>Lys</td><td>Glu</td><td>Phe</td><td>Asp 25</td><td>Glu</td><td>Leu</td><td>Cys</td><td>Phe</td><td>Glu 30</td><td>Tyr</td><td>Gly</td>

<td>Leu</td><td>Glu</td><td>Leu 35</td><td>Asp</td><td>Glu</td><td>ile</td><td>Thr</td><td>Ser 40</td><td>Glu</td><td>Lys</td><td>Ala</td><td>Ala</td><td>Val 45</td><td>Glu</td><td>Lys</td><td>Glu</td>

<td>Gin</td><td>Gly 50</td><td>Thr</td><td>Arg</td><td>Ala</td><td>Ala</td><td>Ser 55</td><td>Asp</td><td>Leu</td><td>Asn</td><td>Asp</td><td>Gin 60</td><td>Glu</td><td>Val</td><td>Tyr</td><td>Lys</td>

<td>Ile 65</td><td>Asp</td><td>Ile</td><td>Pro</td><td>Ala</td><td>Asn 70</td><td>Arg</td><td>Tyr</td><td>Asp</td><td>Leu</td><td>Leu 75</td><td>Ser</td><td>Val</td><td>Glu</td><td>Gly</td><td>Leu 80</td>

<td>Ala</td><td>Arg</td><td>Ala</td><td>Ile</td><td>Arg 85</td><td>Ile</td><td>Phe</td><td>Lys</td><td>Gin</td><td>Glu 90</td><td>Ile</td><td>Pro</td><td>Ser</td><td>Pro</td><td>Ala 95</td><td>Tyr</td>

<td>Lys</td><td>Tyr</td><td>Ala</td><td>Asp 100</td><td>Val</td><td>Pro</td><td>Lys</td><td>Thr</td><td>Gly 105</td><td>Leu</td><td>Gin</td><td>Lys</td><td>Ile</td><td>Ile 110</td><td>Val</td><td>Lys</td>

<td>Ly3</td><td>Glu</td><td>Thr 115</td><td>Ala</td><td>Gin</td><td>Val</td><td>Arg</td><td>Pro 120</td><td>Phe</td><td>Val</td><td>Val</td><td>Gly</td><td>Ala 125</td><td>Val</td><td>Leu</td><td>Arg</td>

<td>Aap</td><td>ile 130</td><td>Ser</td><td>Phe</td><td>Asp</td><td>Ala</td><td>Asp 135</td><td>Ser</td><td>Tyr</td><td>Ala</td><td>Ser</td><td>Phe 140</td><td>Ile</td><td>Asp</td><td>Leu</td><td>Gin</td>

<td>Asp 145</td><td>Lys</td><td>Leu</td><td>His</td><td>Gin</td><td>Asn 150</td><td>Ile</td><td>Cys</td><td>Arg</td><td>Lys</td><td>Arg 155</td><td>Thr</td><td>Leu</td><td>Val</td><td>Ala</td><td>Ile 160</td>

<td>Gly</td><td>Thr</td><td>His</td><td>Asp</td><td>Leu 165</td><td>Asp</td><td>Thr</td><td>Ile</td><td>Gin</td><td>Gly 170</td><td>Pro</td><td>Phe</td><td>Glu</td><td>Tyr</td><td>Arg 175</td><td>Ala</td>

<td>Glu</td><td>Ala</td><td>Pro</td><td>Lys ISO</td><td>Asp</td><td>Ile</td><td>Lys</td><td>Phe</td><td>Lys 185</td><td>Pro</td><td>Leu</td><td>Asn</td><td>Gin</td><td>Thr 190</td><td>Lys</td><td>Glu</td>

<td>Tyr</td><td>Thr</td><td>Ala 195</td><td>Glu</td><td>Glu</td><td>Leu</td><td>Met</td><td>Thr 200</td><td>Leu</td><td>Tyr</td><td>Ser</td><td>Thr</td><td>Asp 205</td><td>Ser</td><td>His</td><td>Leu</td>

<td>Ly3</td><td>Ala 210</td><td>Tyr</td><td>Leu</td><td>Pro</td><td>Ile</td><td>Ile 215</td><td>Gin</td><td>Asn</td><td>His</td><td>Pro</td><td>Val 220</td><td>Tyr</td><td>Pro</td><td>Val</td><td>Ile</td>

<td>Tyr 225</td><td>Asp</td><td>Lys</td><td>Asn</td><td>Gly</td><td>Val 230</td><td>Val</td><td>Cys</td><td>Ser</td><td>Met</td><td>Pro 235</td><td>Pro</td><td>Ile</td><td>Ile</td><td>Asn</td><td>Gly 240</td>

<td>GlU</td><td>His</td><td>Ser</td><td>Lys</td><td>Ile 245</td><td>Thr</td><td>Leu</td><td>Asn</td><td>Thr</td><td>Lys 250</td><td>Asn</td><td>Val</td><td>Phe</td><td>Ile</td><td>Glu 255</td><td>Ala</td>

<td>Thr</td><td>Ala</td><td>Thr</td><td>Asp 260</td><td>Lys</td><td>Gin</td><td>Lys</td><td>Ala</td><td>phe 265</td><td>Val</td><td>Val</td><td>Leu</td><td>Asp</td><td>Thr 270</td><td>Ile</td><td>Val</td>

<td>Thr</td><td>Leu</td><td>Phe 275</td><td>Ser</td><td>Gin</td><td>Tyr</td><td>Cys</td><td>Ala 280</td><td>Lys</td><td>Pio</td><td>Phe</td><td>Thr</td><td>Ile 285</td><td>Glu</td><td>Gin</td><td>v_al</td>

<td>Glu</td><td>Val</td><td>Val</td><td>Tyr</td><td>Glu</td><td>Glu</td><td>Thr</td><td>Gly</td><td>Val</td><td>Lys</td><td>Glu</td><td>Leu</td><td>Tyr</td><td>Pro</td><td>Leu</td><td>Leu</td>

-157-

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>Ser 305</td><td>Tyr</td><td>Arg</td><td>Glu</td><td>Met</td><td>Thr 310</td><td>Val</td><td>Thr</td><td>Thr</td><td>Pro</td><td>Glu 315</td><td>Ile</td><td>Asn</td><td>Thr</td><td>Lys</td><td>Ile 320</td>

<td>Gly</td><td>Ile</td><td>Asn</td><td>Leu</td><td>Lys 325</td><td>Asp</td><td>Glu</td><td>Glu</td><td>Met</td><td>Ala 330</td><td>Thr</td><td>Leu</td><td>Leu</td><td>Asn</td><td>Lys 335</td><td>Met</td>

<td>Ser</td><td>Leu</td><td>Lys</td><td>Ala 340</td><td>Glu</td><td>Val</td><td>Ala</td><td>Ala</td><td>Lys 345</td><td>Glu</td><td>Thr</td><td>Leu</td><td>Lys</td><td>Ile 350</td><td>Val</td><td>val</td>

<td>Pro</td><td>Pro</td><td>Thr 355</td><td>Arg</td><td>His</td><td>Asp</td><td>Ile</td><td>Leu 360</td><td>His</td><td>Ala</td><td>Cys</td><td>Asp</td><td>Ile 365</td><td>Ala</td><td>Glu</td><td>Asp</td>

<td>Val</td><td>Gly 370</td><td>Val</td><td>Ala</td><td>Phe</td><td>Gly</td><td>Tyr 375</td><td>Asn</td><td>Asn</td><td>Leu</td><td>He</td><td>Thr 380</td><td>Lys</td><td>Leu</td><td>Pro</td><td>Glu</td>

<td>Ser 385</td><td>Asn</td><td>Thr</td><td>Val</td><td>Ala</td><td>Val 390</td><td>Ala</td><td>Phe</td><td>Pro</td><td>Ile</td><td>Asn 395</td><td>Ly3</td><td>Leu</td><td>Cys</td><td>Asp</td><td>Asn 400</td>

<td>Leu</td><td>Arg</td><td>Ile</td><td>Glu</td><td>Ile 405</td><td>Ala</td><td>Ala</td><td>Ala</td><td>Gly</td><td>Trp 410</td><td>Thr</td><td>Glu</td><td>Ala</td><td>Leu</td><td>Asn 415</td><td>Phe</td>

<td>Ala</td><td>Leu</td><td>Cys</td><td>Ser 420</td><td>Arg</td><td>Asp</td><td>Asp</td><td>Ile</td><td>Ser 425</td><td>Ser</td><td>Lys</td><td>Leu</td><td>Arg</td><td>Gin 4 30</td><td>Pro</td><td>Asp</td>

<td>Ala</td><td>Leu</td><td>Ser 435</td><td>His</td><td>Ala</td><td>Val</td><td>His</td><td>Ile 440</td><td>Gly</td><td>Asn</td><td>pro</td><td>Lys</td><td>Thr 445</td><td>Leu</td><td>Glu</td><td>Phe</td>

<td>Gin</td><td>Val 450</td><td>Ala</td><td>Arg</td><td>Thr</td><td>Ser</td><td>Leu 455</td><td>Leu</td><td>Pro</td><td>Gly</td><td>Leu</td><td>Leu 460</td><td>Lys</td><td>Thr</td><td>Leu</td><td>Ser</td>

<td>Ser 465</td><td>Asn</td><td>Arg</td><td>Asp</td><td>Met</td><td>Pro 470</td><td>Leu</td><td>Pro</td><td>Leu</td><td>Lys</td><td>Leu 475</td><td>Phe</td><td>Glu</td><td>Leu</td><td>Gin</td><td>Asp 480</td>

<td>Val</td><td>Ile</td><td>Val</td><td>Lys</td><td>Asp 485</td><td>Ser</td><td>Asn</td><td>Thr</td><td>Asp</td><td>Val 4 90</td><td>Gly</td><td>Ala</td><td>Arg</td><td>Asn</td><td>Glu 495</td><td>Arg</td>

<td>Arg</td><td>Leu</td><td>Ala</td><td>Ala 500</td><td>Val</td><td>Tyr</td><td>Tyr</td><td>Asn</td><td>Arg 505</td><td>Ala</td><td>Ala</td><td>Gly</td><td>Phe</td><td>Glu 510</td><td>Ile</td><td>Ile</td>

<td>Gin</td><td>Gly</td><td>Phe 515</td><td>Leu</td><td>Asp</td><td>Arg</td><td>Ile</td><td>Met 520</td><td>Arg</td><td>Met</td><td>Leu</td><td>Asn</td><td>Val 525</td><td>Asn</td><td>Pro</td><td>Ala</td>

<td>Arg</td><td>Asp 530</td><td>Gly</td><td>Thr</td><td>Gly</td><td>Tyr</td><td>Tyr 535</td><td>Ile</td><td>Glu</td><td>Ala</td><td>Asp</td><td>Glu 540</td><td>Asn</td><td>Ser</td><td>Thr</td><td>Tyr</td>

<td>Phe 545</td><td>Pro</td><td>Gly</td><td>Arg</td><td>Cys</td><td>Ala 550</td><td>Lys</td><td>Ile</td><td>Ile</td><td>Gly</td><td>Pro 555</td><td>Lys</td><td>Gly</td><td>Val</td><td>Val</td><td>Leu 560</td>

<td>Gly</td><td>His</td><td>Ile</td><td>Gly</td><td>Ala 565</td><td>Leu</td><td>His</td><td>Pro</td><td>Glu</td><td>Val 570</td><td>Ile</td><td>Thr</td><td>Ser</td><td>Phe</td><td>Gly 575</td><td>Leu</td>

<td>Thr</td><td>Leu</td><td>Pro</td><td>Cys 580</td><td>Gly</td><td>Ala</td><td>Val</td><td>Glu</td><td>Ile 585</td><td>Asn</td><td>Val</td><td>Glu</td><td>Pro</td><td>phe 590</td><td>Leu</td><td></td>

&lt;210&gt; 86 &lt;211&gt; 804 &lt;212&gt; PRT &lt;213&gt; Bacillus subtilis &lt;400&gt; 86

<td>Met</td><td>Phe</td><td>Val</td><td>Ser</td><td>Tyr</td><td>Lys</td><td>Trp</td><td>Leu</td><td>Glu</td><td>Asp</td><td>Tyr</td><td>Val</td><td>Asp</td><td>Leu</td><td>Lys</td><td>Gly</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Met</td><td>Asp</td><td>Pro</td><td>Ala</td><td>Val</td><td>Leu</td><td>Ala</td><td>Glu</td><td>Lys</td><td>Ile</td><td>Thr</td><td>Arg</td><td>Ala</td><td>Gly</td><td>Ile</td><td>Glu</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Val</td><td>Glu</td><td>Gly</td><td>Ile</td><td>Glu</td><td>Tyr</td><td>Lys</td><td>Gly</td><td>Glu</td><td>Gly</td><td>ile</td><td>Lys</td><td>Gly</td><td>Val</td><td>Val</td><td>Ile</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Gly</td><td>His</td><td>Val</td><td>Leu</td><td>Glu</td><td>Arg</td><td>Glu</td><td>Gin</td><td>His</td><td>Pro</td><td>Asn</td><td>Ala</td><td>Asp</td><td>Lys</td><td>Leu</td><td>Asn</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Lys</td><td>Cys</td><td>Leu</td><td>Val</td><td>Asp</td><td>Ile</td><td>Gly</td><td>Ala</td><td>Glu</td><td>Ala</td><td>Pro</td><td>Val</td><td>Gin</td><td>Ile</td><td>Ile</td><td>Cys</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Gly</td><td>Ala</td><td>Pro</td><td>Asn</td><td>Val</td><td>Asp</td><td>Lys</td><td>Gly</td><td>Gin</td><td>Lys</td><td>Val</td><td>Ala</td><td>Val</td><td>Ala</td><td>Thr</td><td>Val</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Gly</td><td>Ala</td><td>Val</td><td>Leu</td><td>Pro</td><td>Gly</td><td>Asn</td><td>Phe</td><td>Lys</td><td>Ile</td><td>Lys</td><td>Lys</td><td>Ala</td><td>Lys</td><td>Leu</td><td>Arg</td>

-158-

<td>Gly</td><td>Glu</td><td>Glu</td><td>100 Ser</td><td>Asn</td><td>Gly</td><td>Met</td><td>Ile</td><td>105 Cys</td><td>Ser</td><td>Leu</td><td>Gin</td><td>Glu</td><td>110 Leu</td><td>Gly</td><td>Ile</td>

<td>Glu</td><td>Ser</td><td>115 Lys</td><td>Leu</td><td>Val</td><td>Ala</td><td>Lys</td><td>120 Glu</td><td>Tyr</td><td>Ala</td><td>Glu</td><td>Gly</td><td>125 Ile</td><td>Phe</td><td>Val</td><td>Phe</td>

<td>Pro</td><td>130 Asn</td><td>Asp</td><td>Ala</td><td>Glu</td><td>Thr</td><td>13 5 Gly</td><td>Ser</td><td>Asp</td><td>Ala</td><td>Leu</td><td>140 Ala</td><td>Ala</td><td>Leu</td><td>Gin</td><td>Leu</td>

<td>14 5 Asp</td><td>Asp</td><td>Ala</td><td>Ile</td><td>Leu</td><td>150 Glu</td><td>Leu</td><td>Gly</td><td>Leu</td><td>Thr</td><td>155 Pro</td><td>Asn</td><td>Arg</td><td>Ala</td><td>Asp</td><td>160 Ala</td>

<td>Met</td><td>Asn</td><td>Met</td><td>Leu</td><td>165 Gly</td><td>Val</td><td>Ala</td><td>Tyr</td><td>Glu</td><td>170 vai</td><td>Ala</td><td>Ala</td><td>Ile</td><td>Leu</td><td>175 Asp</td><td>Thr</td>

<td>Glu</td><td>Val</td><td>Lys</td><td>18 0 Leu</td><td>Pro</td><td>Gin</td><td>Thr</td><td>Asp</td><td>185 Tyr</td><td>Pro</td><td>Ala</td><td>Ala</td><td>Ser</td><td>190 Glu</td><td>Gin</td><td>Ala</td>

<td>.Ser</td><td>Asp</td><td>195 Tyr</td><td>Ile</td><td>Ser</td><td>Val</td><td>Lys</td><td>200 Ile</td><td>Glu</td><td>Asp</td><td>Gin</td><td>Glu</td><td>205 Ala</td><td>Asn</td><td>Pro</td><td>Leu</td>

<td>Tyr</td><td>210 Thr</td><td>Ala</td><td>Lys</td><td>Ile</td><td>ile</td><td>215 Lys</td><td>Asn</td><td>Val</td><td>Thr</td><td>Ile</td><td>220 Ala</td><td>Pro</td><td>Ser</td><td>Pro</td><td>Leu</td>

<td>225 Trp</td><td>Met</td><td>Gin</td><td>Thr</td><td>Lys</td><td>230 Leu</td><td>Met</td><td>Asn</td><td>Ala</td><td>Gly</td><td>235 Ile</td><td>Arg</td><td>Pro</td><td>His</td><td>Asn</td><td>240 Asn</td>

<td>Val</td><td>Val</td><td>Asp</td><td>Ile</td><td>245 Thr</td><td>Asn</td><td>Phe</td><td>Val</td><td>Leu</td><td>250 Leu</td><td>Glu</td><td>Tyr</td><td>Gly</td><td>Gin</td><td>255 Pro</td><td>Leu</td>

<td>His</td><td>Ala</td><td>Phe</td><td>260 Asp</td><td>Tyr</td><td>Asp</td><td>Arg</td><td>Phe</td><td>265 Gly</td><td>Ser</td><td>Lys</td><td>Glu</td><td>Val</td><td>270 Val</td><td>Val</td><td>Arg</td>

<td>Lys</td><td>Ala</td><td>275 Ala</td><td>Glu</td><td>Asn</td><td>Glu</td><td>Met</td><td>280 Ile</td><td>val</td><td>Thr</td><td>Leu</td><td>Asp</td><td>285 Asp</td><td>Gin</td><td>Glu</td><td>Arg</td>

<td>Lys</td><td>290 Leu</td><td>Ser</td><td>Ala</td><td>Asp</td><td>His</td><td>295 Leu</td><td>Val</td><td>Ile</td><td>Thr</td><td>Asn</td><td>300 Gly</td><td>Thr</td><td>Lys</td><td>Ala</td><td>Gin</td>

<td>305 Ala</td><td>Val</td><td>Ala</td><td>Gly</td><td>Val</td><td>310 Met</td><td>Gly</td><td>Gly</td><td>Ala</td><td>Glu</td><td>315 Ser</td><td>Glu</td><td>Val</td><td>Gin</td><td>Glu</td><td>320 Asp</td>

<td>Thr</td><td>Lys</td><td>Thr</td><td>Ile</td><td>325 Leu</td><td>Leu</td><td>Glu</td><td>Ala</td><td>Ala</td><td>330 Tyr</td><td>Phe</td><td>Asn</td><td>Gly</td><td>Gin</td><td>335 Lya</td><td>Val</td>

<td>Arg</td><td>Lys</td><td>Ala</td><td>340 Ser</td><td>Lys</td><td>Asp</td><td>Leu</td><td>Gly</td><td>345 Leu</td><td>Arg</td><td>Ser</td><td>Glu</td><td>Ser</td><td>350 Ser</td><td>Val</td><td>Arg</td>

<td>Phe</td><td>Glu</td><td>355 Lys</td><td>Gly</td><td>Ile</td><td>ASP</td><td>Pro</td><td>360 Ala</td><td>Arg</td><td>val</td><td>Arg</td><td>Leu</td><td>365 Ala</td><td>Ala</td><td>Glu</td><td>Arg</td>

<td>Ala</td><td>370 Ala</td><td>Gin</td><td>Leu</td><td>Ile</td><td>His</td><td>375 Leu</td><td>Tyr</td><td>Ala</td><td>Gly</td><td>Gly</td><td>380 Glu</td><td>Val</td><td>Leu</td><td>Ala</td><td>Gly</td>

<td>385 Thr</td><td>val</td><td>Glu</td><td>Glu</td><td>Asp</td><td>390 His</td><td>Leu</td><td>Thr</td><td>Ile</td><td>Glu</td><td>395 Ala</td><td>Asn</td><td>Asn</td><td>Ile</td><td>His</td><td>400 Val</td>

<td>Ser</td><td>Ala</td><td>Asp</td><td>Lys</td><td>405 Val</td><td>Ser</td><td>Ser</td><td>Val</td><td>Leu</td><td>410 Gly</td><td>Leu</td><td>Thr</td><td>Ile</td><td>Ser</td><td>415 Lys</td><td>Glu</td>

<td>Glu</td><td>Leu</td><td>Ile</td><td>420 Ser</td><td>Ile</td><td>Tyr</td><td>Lys</td><td>Arg</td><td>425 Leu</td><td>Gly</td><td>Phe</td><td>Thr</td><td>Val</td><td>430 Gly</td><td>Glu</td><td>Ala</td>

<td>Asp</td><td>Asp</td><td>435 Leu</td><td>Leu</td><td>val</td><td>vai</td><td>Thr</td><td>440 val</td><td>Pro</td><td>Ser</td><td>Arg</td><td>Arg</td><td>445 Gly</td><td>Asp</td><td>ile</td><td>Thr</td>

<td>Ile</td><td>450 Glu</td><td>Glu</td><td>Asp</td><td>Leu</td><td>Ile</td><td>455 Glu</td><td>Glu</td><td>Ala</td><td>Ala</td><td>Arg</td><td>460 Leu</td><td>Tyr</td><td>Gly</td><td>Tyr</td><td>Asp</td>

<td>465 Asn</td><td>Ile</td><td>Pro</td><td>Ser</td><td>Thr</td><td>470 Leu</td><td>Pro</td><td>Glu</td><td>Thr</td><td>Ala</td><td>475 Gly</td><td>Thr</td><td>Thr</td><td>Gly</td><td>Gly</td><td>480 Leu</td>

<td>Thr</td><td>Pro</td><td>Tyr</td><td>Gin</td><td>485 Ala</td><td>Lys</td><td>Arg</td><td>Arg</td><td>Lys</td><td>490 Val</td><td>Arg</td><td>Arg</td><td>Phe</td><td>Leu</td><td>4 95 Glu</td><td>Gly</td>

<td>Ala</td><td>Gly</td><td>Leu</td><td>500 Ser</td><td>Gin</td><td>Ala</td><td>Ile</td><td>Thr</td><td>505 Tyr</td><td>Ser</td><td>Leu</td><td>Thr</td><td>Asn</td><td>510 Glu</td><td>Lys</td><td>Lys</td>

<td>Ala</td><td>Thr</td><td>515 Ala</td><td>Phe</td><td>Ala</td><td>Ile</td><td>Glu</td><td>520 Lys</td><td>Ser</td><td>Leu</td><td>Asn</td><td>Thr</td><td>525 Val</td><td>Leu</td><td>Ala</td><td>Leu</td>

<td>Pro</td><td>530 Met</td><td>Ser</td><td>Glu</td><td>Glu</td><td>Arg</td><td>535 Ser</td><td>Ile</td><td>Leu</td><td>Arg</td><td>His</td><td>540 Ssr</td><td>Leu</td><td>Wal</td><td>Pro</td><td>Asn</td>

<td>545 Leu</td><td>Leu</td><td>ASp</td><td>Ser</td><td>Val</td><td>550 Ser</td><td>Tyr</td><td>Asn</td><td>Leu</td><td>Ala</td><td>555 Arg</td><td>Gin</td><td>Thr</td><td>Asp</td><td>Ser</td><td>560 Val</td>

5WWWWWJWWW

-159-

<td>Ala</td><td>Leu</td><td>Tyr</td><td>Glu</td><td>565 Val</td><td>Gly</td><td>Ser</td><td>Val</td><td>Phe</td><td>570 Leu</td><td>Thr</td><td>Ly3</td><td>Glu</td><td>Glu</td><td>575 Asp</td><td>Thr</td>

<td>Lya</td><td>Pro</td><td>Val</td><td>580 Glu</td><td>Thr</td><td>Glu</td><td>Arg</td><td>Val</td><td>585 Ala</td><td>Gly</td><td>Ala</td><td>Val</td><td>Thr</td><td>590 Gly</td><td>Leu</td><td>Trp</td>

<td>Arg</td><td>Lys</td><td>595 Gin</td><td>Leu</td><td>Trp</td><td>Gin</td><td>Gly</td><td>600 Glu</td><td>Lys</td><td>Lys</td><td>Pro</td><td>Val</td><td>605 Asp</td><td>Phe</td><td>Phe</td><td>Val</td>

<td>Val</td><td>610 Lys</td><td>Gly</td><td>Ile</td><td>Val</td><td>Glu</td><td>615 Gly</td><td>Leu</td><td>Leu</td><td>Asp</td><td>Lys</td><td>620 Leu</td><td>Asn</td><td>Val</td><td>Leu</td><td>Asp</td>

<td>62 5 Ser</td><td>Ile</td><td>Glu</td><td>Phe</td><td>Val</td><td>630 Gin</td><td>Ser</td><td>Glu</td><td>Arg</td><td>Lys</td><td>635 Gin</td><td>Leu</td><td>His</td><td>Pro</td><td>Gly</td><td>640 Arg</td>

<td>Thr</td><td>Ala</td><td>Asn</td><td>Ile</td><td>645 Leu</td><td>Leu</td><td>Asn</td><td>Gly</td><td>Ser</td><td>650 Leu</td><td>Ile</td><td>Gly</td><td>Phe</td><td>Ile</td><td>655 Gly</td><td>Gin</td>

<td>Val</td><td>His</td><td>Pro</td><td>660 Ser</td><td>Leu</td><td>Glu</td><td>Lys</td><td>Glu</td><td>665 Leu</td><td>Asp</td><td>Ile</td><td>Lys</td><td>Glu</td><td>670 Thr</td><td>Tyr</td><td>Val</td>

<td>Phe</td><td>Glu</td><td>675 Leu</td><td>Asp</td><td>Leu</td><td>His</td><td>Ala</td><td>680 Leu</td><td>Leu</td><td>Ala</td><td>Ala</td><td>Glu</td><td>685 Thr</td><td>Ala</td><td>Pro</td><td>Leu</td>

<td>Val</td><td>690 Tyr</td><td>Thr</td><td>Ala</td><td>Ile</td><td>Pro</td><td>695 Lys</td><td>Tyr</td><td>Pro</td><td>Ser</td><td>Val</td><td>700 Thr</td><td>Arg</td><td>Asp</td><td>Ile</td><td>Ala</td>

<td>705 Leu</td><td>Val</td><td>Thr</td><td>Asp</td><td>Ly3</td><td>710 Thr</td><td>Val</td><td>Thr</td><td>Ser</td><td>Gly</td><td>715 Gin</td><td>Leu</td><td>Glu</td><td>Ser</td><td>val</td><td>720 Ile</td>

<td>Lys</td><td>Glu</td><td>Ala</td><td>Gly</td><td>725 Gly</td><td>Lys</td><td>Leu</td><td>Leu</td><td>Lys</td><td>730 Glu</td><td>Val</td><td>Thr</td><td>Val</td><td>Phe</td><td>735 Asp</td><td>Val</td>

<td>Tyr</td><td>Glu</td><td>Gly</td><td>740 GlU</td><td>His</td><td>Met</td><td>Glu</td><td>Glu</td><td>745 Gly</td><td>Lys</td><td>Lys</td><td>Ser</td><td>Val</td><td>750 Ala</td><td>Phe</td><td>Ser</td>

<td>Leu</td><td>Gin</td><td>755 Tyr</td><td>Val</td><td>Asn</td><td>Pro</td><td>Glu</td><td>760 Gin</td><td>Thr</td><td>Leu</td><td>Thr</td><td>Glu</td><td>765 Glu</td><td>Glu</td><td>Val</td><td>Thr</td>

<td>Lys</td><td>770 Ala</td><td>His</td><td>Ser</td><td>Lys</td><td>Val</td><td>775 Leu</td><td>Lys</td><td>Ala</td><td>Leu</td><td>Glu</td><td>780 Asp</td><td>Thr</td><td>Tyr</td><td>Gin</td><td>Ala</td>

<td>785 Val</td><td>Leu</td><td>Arg</td><td>Gly</td><td></td><td> 790</td><td></td><td></td><td></td><td></td><td> 795</td><td></td><td></td><td></td><td></td><td> 800</td>

&lt;210&gt; 87 &lt;211&gt; 595 &lt;212&gt; PRT &lt;213&gt; Saccharomyces cerevisiae &lt;400&gt; 87

<td>Met 1</td><td>Pro</td><td>Thr</td><td>Val</td><td>Ser 5</td><td>Val</td><td>Asn</td><td>Lys</td><td>Gin</td><td>Gin 10</td><td>Leu</td><td>Phe</td><td>Asp</td><td>Leu</td><td>Leu 15</td><td>Gly</td>

<td>Lys</td><td>Asn</td><td>Tyr</td><td>Thr 20</td><td>Ser</td><td>Gin</td><td>Glu</td><td>Phe</td><td>Asp 25</td><td>Glu</td><td>Leu</td><td>Cys</td><td>Phe</td><td>Glu 30</td><td>Phe</td><td>Gly</td>

<td>Met</td><td>Glu</td><td>Met 35</td><td>Asp</td><td>Glu</td><td>Asp</td><td>Thr</td><td>Thr 40</td><td>Glu</td><td>Glu</td><td>Ala</td><td>Leu</td><td>Lys 45</td><td>Thr</td><td>Gly</td><td>Glu</td>

<td>Glu</td><td>Pro 50</td><td>Glu</td><td>Leu</td><td>Lys</td><td>Leu</td><td>Asp 55</td><td>Ile</td><td>Ser</td><td>Ala</td><td>Asn</td><td>Arg 60</td><td>Tyr</td><td>ASP</td><td>Leu</td><td>Leu</td>

<td>Cys 65</td><td>Ile</td><td>Glu</td><td>Gly</td><td>Ile</td><td>Ser 70</td><td>Gin</td><td>Ser</td><td>Leu</td><td>Asn</td><td>Glu 75</td><td>Tyr</td><td>Leu</td><td>Glu</td><td>Arg</td><td>Lys 80</td>

<td>Glu</td><td>Arg</td><td>Pro</td><td>Asp</td><td>Tyr 85</td><td>Lys</td><td>Leu</td><td>Ser</td><td>Lys</td><td>Pro 90</td><td>Thr</td><td>Thr</td><td>Lys</td><td>Leu</td><td>Ile 95</td><td>Ile</td>

<td>Asp</td><td>Lys</td><td>Ser</td><td>Thr 100</td><td>Glu</td><td>Gin</td><td>Ile</td><td>Arg</td><td>Pro 105</td><td>Phe</td><td>Ala</td><td>Thr</td><td>Ala</td><td>Ala 110</td><td>Val</td><td>Leu</td>

<td>Arg</td><td>Asn</td><td>Ile 115</td><td>Lys</td><td>Leu</td><td>Asn</td><td>Glu</td><td>Lys 120</td><td>Ser</td><td>Tyr</td><td>Ala</td><td>Ser</td><td>Phe 125</td><td>Ile</td><td>Ala</td><td>Leu</td>

<td>Gin</td><td>Asp 13 0</td><td>Lys</td><td>Leu</td><td>His</td><td>Ala</td><td>Asn 135</td><td>Leu</td><td>Cys</td><td>Arg</td><td>Asn</td><td>Arg 140</td><td>Ser</td><td>Leu</td><td>Val</td><td>Ala</td>

<td>Met</td><td>Gly</td><td>Thr</td><td>His</td><td>Asp</td><td>Leu</td><td>Asp</td><td>Ser</td><td>Ile</td><td>Glu</td><td>Gly</td><td>Pro.</td><td>Phe</td><td>His</td><td>Tyr</td><td>Arg</td>

-160-

<td>145 Ala</td><td>Leu</td><td>Pro</td><td>Pro</td><td>Lys</td><td>150 Asp</td><td>Ile</td><td>Lys</td><td>Phe</td><td>Val</td><td>155 Pro</td><td>Leu</td><td>Asn</td><td>Gin</td><td>Thr</td><td>160 Gin</td>

<td>Glu</td><td>Phe</td><td>Thr</td><td>Gly</td><td>165 Asp</td><td>Lys</td><td>Leu</td><td>Ile</td><td>Glu</td><td>170 Phe</td><td>Tyr</td><td>Lys</td><td>Ser</td><td>Pro</td><td>175 Glu</td><td>Gin</td>

<td>Lys</td><td>Asn</td><td>Asn</td><td>180 Ile</td><td>Gly</td><td>Arg</td><td>Tyr</td><td>val</td><td>185 His</td><td>Ile</td><td>Ile</td><td>Glu</td><td>Asp</td><td>190 Ser</td><td>Pro</td><td>Val</td>

<td>Phe</td><td>Pro</td><td>195 Val</td><td>ile</td><td>Met</td><td>Asp</td><td>Ser</td><td>200 Lys</td><td>Asp</td><td>Arg</td><td>Val</td><td>Cys</td><td>205 Ser</td><td>Leu</td><td>Pro</td><td>Pro</td>

<td>Leu</td><td>210 Ile</td><td>Asn</td><td>Ser</td><td>Glu</td><td>His</td><td>215 Ser</td><td>Lys</td><td>Ile</td><td>Ser</td><td>Val</td><td>220 Asn</td><td>Thr</td><td>Arg</td><td>Asn</td><td>Ile</td>

<td>225 Leu</td><td>Ile</td><td>Asp</td><td>Ile</td><td>Thr</td><td>230 Ala</td><td>Thr</td><td>Asp</td><td>Lys</td><td>Thr</td><td>235 Lys</td><td>Ala</td><td>Glu</td><td>Ile</td><td>Val</td><td>240 Leu</td>

<td>Asn</td><td>Ile</td><td>Leu</td><td>Thr</td><td>245 Thr</td><td>Met</td><td>Phe</td><td>ser</td><td>Arg</td><td>250 Tyr</td><td>Cys</td><td>ASp</td><td>Glu</td><td>Pro</td><td>255 Phe</td><td>Thr</td>

<td>Val</td><td>Glu</td><td>Pro</td><td>260 Val</td><td>Glu</td><td>Ile</td><td>Val</td><td>Ser</td><td>265 Glu</td><td>His</td><td>Asn</td><td>Gly</td><td>Gin</td><td>270 Ser</td><td>Arg</td><td>Leu</td>

<td>Ala</td><td>Pro</td><td>275 Asn</td><td>Phe</td><td>Asn</td><td>Asp</td><td>Arg</td><td>280 Ile</td><td>Met</td><td>Asp</td><td>Val</td><td>Ser</td><td>285 Ile</td><td>Lys</td><td>Tyr</td><td>Ile</td>

<td>Asn</td><td>290 Ser</td><td>Cys</td><td>Leu</td><td>Gly</td><td>Leu</td><td>295 Asp</td><td>Gin</td><td>Ser</td><td>Ala</td><td>Asp</td><td>300 Glu</td><td>Ile</td><td>Ala</td><td>His</td><td>Cys</td>

<td>305 Leu</td><td>Lys</td><td>Lys</td><td>Met</td><td>Ser</td><td>310 Leu</td><td>His</td><td>Ala</td><td>Val</td><td>Gin</td><td>315 Ser</td><td>Lys</td><td>Glu</td><td>Asp</td><td>Ly3</td><td>320 Asp</td>

<td>Ile</td><td>Leu</td><td>His</td><td>Val</td><td>325 ASp</td><td>Ile</td><td>Pro</td><td>Val</td><td>Thr</td><td>330 Arg</td><td>Pro</td><td>Asp</td><td>Ile</td><td>Leu</td><td>335 His</td><td>Ala</td>

<td>Cy3</td><td>Asp</td><td>Ile</td><td>340 Met</td><td>Glu</td><td>Asp</td><td>Ala</td><td>Ala</td><td>345 Val</td><td>Gly</td><td>Tyr</td><td>Gly</td><td>Phe</td><td>350 Asn</td><td>Asn</td><td>Leu</td>

<td>Pro</td><td>Lys</td><td>355 Gly</td><td>Glu</td><td>Lys</td><td>Leu</td><td>Ser</td><td>360 Asn</td><td>Ala</td><td>Asn</td><td>Phe</td><td>Ile</td><td>365 Ala</td><td>Lys</td><td>Pro</td><td>Leu</td>

<td>Pro</td><td>370 Ile</td><td>Asn</td><td>Lys</td><td>Val</td><td>Ser</td><td>375 Asp</td><td>Ile</td><td>Phe</td><td>Arg</td><td>Val</td><td>380 Ala</td><td>Ser</td><td>Ser</td><td>Gin</td><td>Ala</td>

<td>3 85 Thr</td><td>Trp</td><td>Val</td><td>Glu</td><td>Val</td><td>390 Leu</td><td>Pro</td><td>Leu</td><td>Thr</td><td>Leu</td><td>395 cys</td><td>Ser</td><td>His</td><td>Asp</td><td>Glu</td><td>400 Asn</td>

<td>Phe</td><td>Ly3</td><td>Phe</td><td>Leu</td><td>405 Arg</td><td>Gin</td><td>Ser</td><td>Asp</td><td>Asn</td><td>410 Gly</td><td>Asp</td><td>Leu</td><td>Ala</td><td>Val</td><td>415 Lys</td><td>Leu</td>

<td>Ala</td><td>Asn</td><td>Pro</td><td>420 Lys</td><td>Thr</td><td>Leu</td><td>Glu</td><td>Tyr</td><td>425 Gin</td><td>Val</td><td>Val</td><td>Arg</td><td>Thr</td><td>430 Thr</td><td>Leu</td><td>Leu</td>

<td>Pro</td><td>Gly</td><td>435 Ile</td><td>Leu</td><td>Lys</td><td>Thr</td><td>Val</td><td>440 Lys</td><td>Glu</td><td>Asn</td><td>Arg</td><td>Lys</td><td>445 His</td><td>Ser</td><td>Leu</td><td>pro</td>

<td>Ile</td><td>450 Lys</td><td>Val</td><td>Phe</td><td>Glu</td><td>Thr</td><td>455 Gly</td><td>Asp</td><td>Val</td><td>Val</td><td>Phe</td><td>460 Lys</td><td>Asp</td><td>Asp</td><td>Lys</td><td>Leu</td>

<td>465 Glu</td><td>Arg</td><td>Lys</td><td>Ala</td><td>Tyr</td><td>470 Asn</td><td>Glu</td><td>Arg</td><td>His</td><td>Trp</td><td>475 Ala</td><td>Ala</td><td>Ile</td><td>Tyr</td><td>Val</td><td>4 80 Gly</td>

<td>Lys</td><td>Asn</td><td>Ser</td><td>Gly</td><td>485 Phe</td><td>Glu</td><td>Ile</td><td>Ile</td><td>Gin</td><td>490 Gly</td><td>Leu</td><td>Leu</td><td>Gly</td><td>Lys</td><td>495 Ile</td><td>Met</td>

<td>Gin</td><td>Thr</td><td>Phe</td><td>500 Arg</td><td>Thr</td><td>Glu</td><td>Trp</td><td>Ile</td><td>505 Ala</td><td>Asp</td><td>Tyr</td><td>Gly</td><td>Ala</td><td>510 Ala</td><td>Ala</td><td>Ser</td>

<td>Gly</td><td>Arg</td><td>515 Gly</td><td>Tyr</td><td>Trp</td><td>Ile</td><td>Glu</td><td>520 Glu</td><td>Asp</td><td>Asp</td><td>Ser</td><td>Val</td><td>525 Lys</td><td>Thr</td><td>Tyr</td><td>Phe</td>

<td>Pro</td><td>530 Gly</td><td>Arg</td><td>Gly</td><td>Ala</td><td>Lys</td><td>535 Val</td><td>Met</td><td>Phe</td><td>Arg</td><td>Ser</td><td>540 Lys</td><td>Glu</td><td>Gly</td><td>Ala</td><td>Glu</td>

<td>54 5 Pro</td><td>Lys</td><td>Gin</td><td>Ile</td><td>Gly</td><td>550 His</td><td>Leu</td><td>Gly</td><td>Val</td><td>Leu</td><td>555 His</td><td>Pro</td><td>Glu</td><td>Val</td><td>Met</td><td>560 Met</td>

<td>Asn</td><td>Phe</td><td>Asp</td><td>Val</td><td>565 Pro</td><td>Phe</td><td>Ala</td><td>Ala</td><td>Ser</td><td>570 Phe</td><td>Val</td><td>Glu</td><td>Val</td><td>Asn</td><td>575 Ala</td><td>Glu</td>

580 585 590

Val Phe Leu 595 &lt;210&gt; 88 &lt;211&gt; 792 &lt;212&gt; PRT &lt;213&gt; Chlamydophila pneumoniae &lt;400&gt; 88

-161-

<td>Met 1</td><td>Arg</td><td>Ile</td><td>Pro</td><td>Ile 5</td><td>Thr</td><td>Leu</td><td>Leu</td><td>Gln</td><td>Thr 10</td><td>Tyr</td><td>Phe</td><td>Ser</td><td>Glu</td><td>Pro 15</td><td>Leu</td>

<td>ser</td><td>Thr</td><td>Lys</td><td>Glu 20</td><td>Ile</td><td>Leu</td><td>Glu</td><td>Ala</td><td>Cys 25</td><td>ASp</td><td>His</td><td>Ile</td><td>Gly</td><td>Ile 30</td><td>Glu</td><td>Ala</td>

<td>GlU</td><td>Ile</td><td>Glu 35</td><td>Asn</td><td>Thr</td><td>Thr</td><td>Leu</td><td>Tyr 40</td><td>Ser</td><td>Phe</td><td>Ala</td><td>Ser</td><td>Val 45</td><td>Ile</td><td>Thr</td><td>Ala</td>

<td>Lya</td><td>Ile 50</td><td>Leu</td><td>His</td><td>Thr</td><td>Ile</td><td>Pro 55</td><td>His</td><td>Pro</td><td>Asn</td><td>Ala</td><td>Asp 60</td><td>Lys</td><td>Leu</td><td>Arg</td><td>Val</td>

<td>Ala 65</td><td>Thr</td><td>Leu</td><td>Thr</td><td>Asp</td><td>Gly 70</td><td>Glu</td><td>Lys</td><td>Glu</td><td>His</td><td>Gln 75</td><td>Val</td><td>Val</td><td>Cys</td><td>Gly</td><td>Ala 80</td>

<td>Pro</td><td>Asn</td><td>Cys</td><td>Glu</td><td>Ala 85</td><td>Gly</td><td>Leu</td><td>Ile</td><td>Val</td><td>Ala 90</td><td>Leu</td><td>Ala</td><td>Leu</td><td>Pro</td><td>Gly 95</td><td>Ala</td>

<td>Lys</td><td>Leu</td><td>Phe</td><td>Asp 100</td><td>Ser</td><td>Glu</td><td>Gly</td><td>Gln</td><td>Ala 105</td><td>Tyr</td><td>Thr</td><td>Ile</td><td>Lys</td><td>Lys 110</td><td>Ser</td><td>Lys</td>

<td>Leu</td><td>Arg</td><td>Gly 115</td><td>Val</td><td>Glu</td><td>Ser</td><td>Gln</td><td>Gly 120</td><td>Met</td><td>Cys</td><td>Cys</td><td>Gly</td><td>Ala 125</td><td>Asp</td><td>Glu</td><td>Leu</td>

<td>Gly</td><td>Leu 130</td><td>Asp</td><td>Glu</td><td>Leu</td><td>Gln</td><td>Ile 135</td><td>Gln</td><td>Glu</td><td>Arg</td><td>Ala</td><td>Leu 140</td><td>Leu</td><td>Glu</td><td>Leu</td><td>Pro</td>

<td>Glu 14 5</td><td>Ala</td><td>Thr</td><td>Pro</td><td>Leu</td><td>Gly 150</td><td>Glu</td><td>Asp</td><td>Leu</td><td>Ala</td><td>Thr 155</td><td>Val</td><td>Leu</td><td>Gly</td><td>Asn</td><td>Thr 160</td>

<td>Ser</td><td>Leu</td><td>Glu</td><td>Ile</td><td>Ser 165</td><td>Leu</td><td>Thr</td><td>Pro</td><td>Asn</td><td>Leu 170</td><td>Gly</td><td>His</td><td>Cys</td><td>Ala</td><td>Ser 175</td><td>Phe</td>

<td>Leu</td><td>Gly</td><td>Leu</td><td>Ala 180</td><td>Arg</td><td>Glu</td><td>He</td><td>Cys</td><td>His 185</td><td>Val</td><td>Thr</td><td>Gln</td><td>Ala</td><td>Asn 190</td><td>Leu</td><td>Val</td>

<td>Ile</td><td>Pro</td><td>Lys 195</td><td>Glu</td><td>Phe</td><td>Ser</td><td>Phe</td><td>Glu 200</td><td>Asn</td><td>Leu</td><td>Pro</td><td>Thr</td><td>Thr 205</td><td>Ala</td><td>Leu</td><td>Asp</td>

<td>Met</td><td>Gly 210</td><td>Asn</td><td>Asp</td><td>Pro</td><td>Asp</td><td>ile 215</td><td>Cys</td><td>Pro</td><td>Phe</td><td>Phe</td><td>Ser 220</td><td>Tyr</td><td>Val</td><td>Val</td><td>Ile</td>

<td>Thr 225</td><td>Gly</td><td>Ile</td><td>Ser</td><td>Ala</td><td>Gln 230</td><td>Pro</td><td>Ser</td><td>Pro</td><td>Ile</td><td>Lys 235</td><td>Leu</td><td>Gln</td><td>Glu</td><td>Ser</td><td>Leu 240</td>

<td>Gln</td><td>Ala</td><td>Leu</td><td>Lys</td><td>Gln 245</td><td>Lys</td><td>Pro</td><td>Ile</td><td>Asn</td><td>Ala 250</td><td>Ile</td><td>Val</td><td>Asp</td><td>Ile</td><td>Thr 255</td><td>Asn</td>

<td>Tyr</td><td>Ile</td><td>Met</td><td>Leu 260</td><td>Ser</td><td>Leu</td><td>Gly</td><td>Gln</td><td>Pro 265</td><td>Leu</td><td>His</td><td>Ala</td><td>Tyr</td><td>Asp 270</td><td>Ala</td><td>Ser</td>

<td>His</td><td>Val</td><td>Ala 275</td><td>Leu</td><td>Asp</td><td>Ser</td><td>Leu</td><td>Arg 280</td><td>Val</td><td>Glu</td><td>Lys</td><td>Leu</td><td>Ser 285</td><td>Thr</td><td>Pro</td><td>Glu</td>

<td>Ser</td><td>Leu 290</td><td>Thr</td><td>Leu</td><td>Leu</td><td>Asn</td><td>Gly 295</td><td>Glu</td><td>Thr</td><td>val</td><td>Leu</td><td>Leu 300</td><td>Pro</td><td>Ser</td><td>Gly</td><td>Val</td>

<td>Pro 305</td><td>Val</td><td>Val</td><td>Arg</td><td>Asp</td><td>Asp 310</td><td>His</td><td>Ser</td><td>Leu</td><td>Leu</td><td>Gly 315</td><td>Leu</td><td>Gly</td><td>Gly</td><td>Val</td><td>Met 320</td>

<td>Gly</td><td>Ala</td><td>Lys</td><td>Ala</td><td>Pro 325</td><td>Ser</td><td>Phe</td><td>Gln</td><td>Glu</td><td>Thr 330</td><td>Thr</td><td>Thr</td><td>Thr</td><td>Thr</td><td>Val 335</td><td>Ile</td>

<td>Lys</td><td>Ala</td><td>Ala</td><td>Tyr 340</td><td>Phe</td><td>Leu</td><td>Pro</td><td>Glu</td><td>Ala 345</td><td>Leu</td><td>Arg</td><td>Ala</td><td>Ser</td><td>Gln 350</td><td>Lys</td><td>Leu</td>

<td>Leu</td><td>Pro</td><td>Ile 355</td><td>Pro</td><td>Ser</td><td>Glu</td><td>Ser</td><td>Ala 360</td><td>Tyr</td><td>Arg</td><td>Phe</td><td>Thr</td><td>Arg 365</td><td>Gly</td><td>Ile</td><td>Asp</td>

<td>Pro</td><td>Gln 370</td><td>Asn</td><td>Val</td><td>Val</td><td>Pro</td><td>Ala 375</td><td>Leu</td><td>Gln</td><td>Ala</td><td>Ala</td><td>Ile 380</td><td>His</td><td>Tyr</td><td>Ile</td><td>Leu</td>

<td>Glu 385</td><td>Ile</td><td>Phe</td><td>Pro</td><td>Glu</td><td>Ala 390</td><td>Thr</td><td>Ile</td><td>Ser</td><td>Pro</td><td>Ile 395</td><td>Tyr</td><td>Ser</td><td>ser</td><td>Gly</td><td>Glu 400</td>

<td>Ile</td><td>Cys</td><td>Arg</td><td>Glu</td><td>Leu</td><td>Lys</td><td>Glu</td><td>Val</td><td>Ala</td><td>Leu</td><td>Arg</td><td>Pro</td><td>Lys</td><td>Thr</td><td>Leu</td><td>Gln</td>

-162-

<td>Arg</td><td>Ile</td><td>Leu</td><td>Gly</td><td>405 Lys</td><td>Ser</td><td>Phe</td><td>Ser</td><td>Ile</td><td>410 Glu</td><td>Ile</td><td>Leu</td><td>Ser</td><td>Gin</td><td>415 Łys</td><td>Leu.</td>

<td>Gin</td><td>Ser</td><td>Leu</td><td>420 Gly</td><td>Phe</td><td>Ser</td><td>Thr</td><td>Thr</td><td>425 Pro</td><td>Gin</td><td>Glu</td><td>Thr</td><td>Ser</td><td>430 Leu</td><td>Leu</td><td>val</td>

<td>Lys</td><td>Val</td><td>435 Pro</td><td>Ser</td><td>Tyr</td><td>Arg</td><td>His</td><td>440 Asp</td><td>Ile</td><td>Asn</td><td>Glu</td><td>Glu</td><td>445 Ile</td><td>Asp</td><td>Leu</td><td>Val</td>

<td>Glu</td><td>450 Glu</td><td>Ile</td><td>Cys</td><td>Arg</td><td>Thr</td><td>455 Glu</td><td>Ser</td><td>Trp</td><td>Asn</td><td>Ile</td><td>460 Glu</td><td>Thr</td><td>Gin</td><td>Asn</td><td>Pro</td>

<td>465 Val</td><td>Ser</td><td>Cys</td><td>Tyr</td><td>Thr</td><td>470 Pro</td><td>Ile</td><td>Tyr</td><td>Lys</td><td>Leu</td><td>475 Lys</td><td>Arg</td><td>Glu</td><td>Thr</td><td>Ala</td><td>480 Gly</td>

<td>Phe</td><td>Leu</td><td>Ala</td><td>Asn</td><td>485 Ala</td><td>Gly</td><td>Leu</td><td>Gin</td><td>Glu</td><td>490 Phe</td><td>Phe</td><td>Thr</td><td>Pro</td><td>Asp</td><td>495 Leu</td><td>Leu</td>

<td>Asp</td><td>Pro</td><td>Glu</td><td>500 Thr</td><td>Val</td><td>Ala</td><td>Leu</td><td>Thr</td><td>505 Arg</td><td>Lys</td><td>Glu</td><td>LyS</td><td>Glu</td><td>510 Glu</td><td>Ile</td><td>Ser</td>

<td>Leu</td><td>Gin</td><td>515 Gly</td><td>Ser</td><td>Lys</td><td>His</td><td>Thr</td><td>520 Thr</td><td>val</td><td>Leu</td><td>Arg</td><td>Ser</td><td>525 Ser</td><td>Leu</td><td>Leu</td><td>Pro</td>

<td>Gly</td><td>530 Leu</td><td>Leu</td><td>Lys</td><td>Ser</td><td>Ala</td><td>535 Ala</td><td>Thr</td><td>Asn</td><td>Leu</td><td>Asn</td><td>540 Arg</td><td>Gin</td><td>Ala</td><td>Pro</td><td>Ser</td>

<td>545 Val</td><td>Gin</td><td>Ala</td><td>Phe</td><td>Glu</td><td>550 Ile</td><td>Gly</td><td>Thr</td><td>Val</td><td>Tyr</td><td>555 Ala</td><td>Lys</td><td>His</td><td>Gly</td><td>Glu</td><td>560 Gin</td>

<td>.Cys</td><td>Gin</td><td>Glu</td><td>Thr</td><td>565 Gin</td><td>Thr</td><td>Leu</td><td>Ala</td><td>Ile</td><td>570 Leu</td><td>Leu</td><td>Thr</td><td>Glu</td><td>Asp</td><td>575 Gly</td><td>Glu</td>

<td>Ser</td><td>Arg</td><td>Ser</td><td>580 Trp</td><td>Leu</td><td>Pro</td><td>Lys</td><td>Pro</td><td>585 Ser</td><td>Leu</td><td>ser</td><td>Phe</td><td>Tyr</td><td>590 Ser</td><td>Leu</td><td>Lys</td>

<td>Gly</td><td>Trp</td><td>595 Val</td><td>Glu</td><td>Arg</td><td>Leu</td><td>Leu</td><td>600 Tyr</td><td>His</td><td>His</td><td>His</td><td>Leu</td><td>605 Ser</td><td>He</td><td>Asp</td><td>Ala</td>

<td>Leu</td><td>610 Thr</td><td>Leu</td><td>Glu</td><td>Ser</td><td>Ser</td><td>615 Ala</td><td>Leu</td><td>Cys</td><td>Glu</td><td>Phe</td><td>620 His</td><td>Pro</td><td>Tyr</td><td>Gin</td><td>Gin</td>

<td>625 Gly</td><td>Val</td><td>Leu</td><td>Arg</td><td>Ile</td><td>630 His</td><td>Lys</td><td>Gin</td><td>Ser</td><td>Phe</td><td>635 Ala</td><td>Thr</td><td>Leu</td><td>Gly</td><td>Gin</td><td>640 Val</td>

<td>His</td><td>Pro</td><td>Glu</td><td>Leu</td><td>645 Ala</td><td>Lys</td><td>Lys</td><td>Ala</td><td>Gin</td><td>650 Ile</td><td>Lys</td><td>Hia</td><td>Pro</td><td>Val</td><td>655 Phe</td><td>Phe</td>

<td>Ala</td><td>Glu</td><td>Leu</td><td>660 Asn.</td><td>Leu</td><td>Asp</td><td>Leu</td><td>Leu</td><td>665 Cys</td><td>Lys</td><td>Met</td><td>Leu</td><td>Lys</td><td>670 Lys</td><td>Thr</td><td>Thr</td>

<td>Lys</td><td>Leu</td><td>675 Tyr</td><td>Lys</td><td>Pro</td><td>Tyr</td><td>Ala</td><td>680 Ile</td><td>Tyr</td><td>Pro</td><td>Ser</td><td>Ser</td><td>68S Phe</td><td>Arg</td><td>Asp</td><td>Leu</td>

<td>Thr</td><td>690 Leu</td><td>Thr</td><td>Val</td><td>Pro</td><td>Glu</td><td>695 ASp</td><td>ile</td><td>Pro</td><td>Ala</td><td>Asn</td><td>700 Leu</td><td>Leu</td><td>Arg</td><td>Gin</td><td>Lys</td>

<td>705 Leu</td><td>Leu</td><td>His</td><td>Glu</td><td>Gly</td><td>710 Ser</td><td>Lys</td><td>Trp</td><td>Leu</td><td>Glu</td><td>715 Ser</td><td>Val</td><td>Thr</td><td>Ile</td><td>Ile</td><td>720 Ser</td>

<td>Ile</td><td>Tyr</td><td>Gin</td><td>Asp</td><td>725 Lys</td><td>Ser</td><td>Leu</td><td>Glu</td><td>Thr</td><td>730 Arg</td><td>Asri</td><td>Lys</td><td>Asn</td><td>Val</td><td>735 Ser</td><td>Leu</td>

<td>Arg</td><td>Leu</td><td>val</td><td>740 Phe</td><td>Gin</td><td>Asp</td><td>Tyr</td><td>Glu</td><td>745 Arg</td><td>Thr</td><td>Leu</td><td>Ser</td><td>Asn</td><td>750 Gin</td><td>Asp</td><td>Ile</td>

<td>Glu</td><td>Glu</td><td>755 Glu</td><td>Tyr</td><td>Cys</td><td>Arg</td><td>Leu</td><td>750 Val</td><td>Ala</td><td>Leu</td><td>Leu</td><td>Asn</td><td>765 Glu</td><td>Leu</td><td>Leu</td><td>Thr</td>

<td>Asp 785</td><td>770 Thr</td><td>Lys</td><td>Gly</td><td>Thr</td><td>Ile 790</td><td>775 Asn</td><td>Ser</td><td></td><td></td><td></td><td> 780</td><td></td><td></td><td></td><td></td>

&lt;210&gt; 89 &lt;211&gt; 831 &lt;212&gt; PRT &lt;213&gt; Mycobacterium tuberculosis &lt;400&gt; 89

Met Arg Leu Pro Tyr Ser Trp Leu Arg Glu Val Val Ala Val Gly Ala

-163

<td>1 Ser</td><td>Gly</td><td>Trp</td><td>Asp</td><td>5 Val</td><td>Thr</td><td>Pro</td><td>Gly</td><td>Glu</td><td>10 Leu</td><td>Glu</td><td>Gin</td><td>Thr</td><td>Leu</td><td>15 Leu</td><td>Arg</td>

<td>Ile</td><td>Gly</td><td>His</td><td>20 Glu</td><td>Val</td><td>Glu</td><td>Glu</td><td>Val</td><td>25 Ile</td><td>Pro</td><td>Leu</td><td>Gly</td><td>Pro</td><td>30 Val</td><td>Asp</td><td>Gly</td>

<td>Pro</td><td>val</td><td>35 Thr</td><td>Val</td><td>Gly</td><td>Arg</td><td>Val</td><td>40 Ala</td><td>Asp</td><td>Ile</td><td>Glu</td><td>Glu</td><td>45 Leu</td><td>Thr</td><td>Gly</td><td>Tyr</td>

<td>Lys</td><td>50 Lys</td><td>Pro</td><td>Ile</td><td>Arg</td><td>Ala</td><td>55 Cys</td><td>Ala</td><td>Val</td><td>Asp</td><td>Ile</td><td>60 Gly</td><td>Asp</td><td>Arg</td><td>Gin</td><td>Tyr</td>

<td>65 Arg</td><td>Glu</td><td>Ile</td><td>Ile</td><td>Cys</td><td>70 Gly</td><td>Ala</td><td>Thr</td><td>Asn</td><td>Phe</td><td>75 Ala</td><td>Val</td><td>Gly</td><td>Asp</td><td>Leu</td><td>80 val</td>

<td>Val</td><td>Val</td><td>Ala</td><td>Leu</td><td>85 Pro</td><td>Gly</td><td>Ala</td><td>Thr</td><td>Leu</td><td>90 Pro</td><td>Gly</td><td>Gly</td><td>Phe</td><td>Thr</td><td>95 Ile</td><td>Ser</td>

<td>Ala</td><td>Arg</td><td>Lys</td><td>100 Ala</td><td>Tyr</td><td>Gly</td><td>Arg</td><td>Asn</td><td>105 Ser</td><td>Asp</td><td>Gly</td><td>Met</td><td>Ile</td><td>110 Cys</td><td>Ser</td><td>Ala</td>

<td>Ala</td><td>Glu</td><td>115 Leu</td><td>Asn</td><td>Leu</td><td>Gly</td><td>Ala</td><td>120 Asp</td><td>His</td><td>Ser</td><td>Gly</td><td>Ile</td><td>12 5 Leu</td><td>val</td><td>Leu</td><td>Pro</td>

<td>Pro</td><td>13 0 Gly</td><td>Ala</td><td>Ala</td><td>Glu</td><td>Pro</td><td>135 Gly</td><td>Ala</td><td>Asp</td><td>Gly</td><td>Ala</td><td>140 Gly</td><td>Val</td><td>Leu</td><td>Gly</td><td>Leu</td>

<td>145 Asp</td><td>Asp</td><td>Val</td><td>Val</td><td>Phe</td><td>¹⁵⁰His</td><td>Leu</td><td>Ala</td><td>Ile</td><td>Thr</td><td>155 Pro</td><td>ASp</td><td>Arg</td><td>Gly</td><td>Tyr</td><td>160 Cys</td>

<td>Met</td><td>Ser</td><td>Val</td><td>Arg</td><td>165 Gly</td><td>Leu</td><td>Ala</td><td>Arg</td><td>Glu</td><td>170 Leu</td><td>Ala</td><td>Cys</td><td>Ala</td><td>Tyr</td><td>17 5 Asp</td><td>Leu</td>

<td>Asp</td><td>Phe</td><td>Val</td><td>180 Asp</td><td>Pro</td><td>Ala</td><td>Ser</td><td>Asn</td><td>185 Ser</td><td>Arg</td><td>Val</td><td>Pro</td><td>Pro</td><td>190 Leu</td><td>Pro</td><td>Ile</td>

<td>Glu</td><td>Gly</td><td>195 Pro</td><td>Ala</td><td>Trp</td><td>Pro</td><td>Leu</td><td>200 Thr</td><td>Val</td><td>Gin</td><td>Pro</td><td>Glu</td><td>205 Thr</td><td>Gly</td><td>Val</td><td>Arg</td>

<td>Arg</td><td>210 Phe</td><td>Ala</td><td>Leu</td><td>Arg</td><td>Pro</td><td>215 Val</td><td>Ile</td><td>Gly</td><td>Ile</td><td>Asp</td><td>220 Pro</td><td>Ala</td><td>Ala</td><td>Val</td><td>Ser</td>

<td>225 Pro</td><td>Trp</td><td>Trp</td><td>Leu</td><td>Gin</td><td>230 Arg</td><td>Arg</td><td>Leu</td><td>Leu</td><td>Leu</td><td>23S Cys</td><td>Gly</td><td>Ile</td><td>Arg</td><td>Ala</td><td>240 Thr</td>

<td>Cys</td><td>Pro</td><td>Ala</td><td>Val</td><td>245 Asp</td><td>Val</td><td>Thr</td><td>Asn</td><td>Tyr</td><td>250 Val</td><td>Met</td><td>Leu</td><td>Glu</td><td>Leu</td><td>2SS Gly</td><td>His</td>

<td>Pro</td><td>Met</td><td>His</td><td>260 Ala</td><td>His</td><td>Asp</td><td>Arg</td><td>Asn</td><td>265 Arg</td><td>Ile</td><td>Ser</td><td>Gly</td><td>'Thr</td><td>270 Leu</td><td>Gly</td><td>Val</td>

<td>Arg</td><td>Phe</td><td>275 Ala</td><td>Arg</td><td>Ser</td><td>Gly</td><td>Glu</td><td>280 Thr</td><td>Ala</td><td>val</td><td>Thr</td><td>Leu</td><td>285 Asp</td><td>Gly</td><td>Ile</td><td>Glu</td>

<td>Arg</td><td>290 Lys</td><td>Leu</td><td>Asp</td><td>Thr</td><td>Ala</td><td>295 Asp</td><td>Val</td><td>Leu</td><td>Ile</td><td>Val</td><td>300 ASp</td><td>Asp</td><td>Ala</td><td>Ala</td><td>Thr</td>

<td>305 Ala</td><td>Ala</td><td>Ile</td><td>Gly</td><td>Gly</td><td>310 Val</td><td>Met</td><td>Gly</td><td>Ala</td><td>Ala</td><td>315 Ser</td><td>Thr</td><td>Glu</td><td>Val</td><td>Arg</td><td>320 Ala</td>

<td>Asp</td><td>Ser</td><td>Thr</td><td>Asp</td><td>325 Val</td><td>Leu</td><td>Leu</td><td>Glu</td><td>Ala</td><td>330 Ala</td><td>Ile</td><td>Trp</td><td>Asp</td><td>Pro</td><td>335 Ala</td><td>Ala</td>

<td>Val</td><td>Ser</td><td>Arg</td><td>340 Thr</td><td>Gin</td><td>Arg</td><td>Arg</td><td>Leu</td><td>345 His</td><td>Leu</td><td>Pro</td><td>Ser</td><td>Glu</td><td>350 Ala</td><td>Ala</td><td>Arg</td>

<td>Arg</td><td>Tyr</td><td>355 Glu</td><td>Arg</td><td>Thr</td><td>Val</td><td>Asp</td><td>360 Pro</td><td>Ala</td><td>Ile</td><td>Ser</td><td>Val</td><td>365 Ala</td><td>Ala</td><td>Leu</td><td>ASp</td>

<td>Arg</td><td>370 Cys</td><td>Ala</td><td>Arg</td><td>Leu</td><td>Leu</td><td>375 Ala</td><td>Asp</td><td>Ile</td><td>Ala</td><td>Gly</td><td>380 Gly</td><td>Glu</td><td>Val</td><td>Ser</td><td>Pro</td>

<td>385 Thr</td><td>Leu</td><td>Thr</td><td>Asp</td><td>Trp</td><td>390 Arg</td><td>Gly</td><td>Asp</td><td>Pro</td><td>pro</td><td>395 Cys</td><td>Asp</td><td>Asp</td><td>Trp</td><td>Ser</td><td>400 Pro</td>

<td>Pro</td><td>Pro</td><td>Ile</td><td>Arg</td><td>405 Met</td><td>Gly</td><td>val</td><td>Asp</td><td>val</td><td>410 Pro</td><td>Asp</td><td>Arg</td><td>Ile</td><td>Ala</td><td>415 Gly</td><td>Val</td>

<td>Ala</td><td>Tyr</td><td>Pro</td><td>420 Gin</td><td>Gly</td><td>Thr</td><td>Thr</td><td>Ala</td><td>425 Arg</td><td>Arg</td><td>Leu</td><td>Ala</td><td>Gin</td><td>430 Ile</td><td>Gly</td><td>Ala</td>

<td>val</td><td>vai</td><td>435 Thr</td><td>His</td><td>Asp</td><td>Gly</td><td>Asp</td><td>440 Thr</td><td>Leu</td><td>Thr</td><td>Val</td><td>Thr</td><td>445 Pro</td><td>Pro</td><td>Ser</td><td>Trp</td>

<td>Arg</td><td>450 Pro</td><td>Asp</td><td>Leu</td><td>Arg</td><td>Gin</td><td>455 Pro</td><td>Ala</td><td>Asp</td><td>Leu</td><td>Val</td><td>450' Glu</td><td>Glu</td><td>Val</td><td>Leu</td><td>Arg</td>

-164-

<td> 465</td><td></td><td></td><td></td><td></td><td> 470</td><td></td><td></td><td></td><td></td><td> 475</td><td></td><td></td><td></td><td></td><td> 480</td>

<td>Leu</td><td>Glu</td><td>Gly</td><td>Leu</td><td>Glu 485</td><td>Val</td><td>Ile</td><td>Pro</td><td>Ser</td><td>Val 490</td><td>Leu</td><td>Pro</td><td>Pro</td><td>Ala</td><td>pro 495</td><td>Ala</td>

<td>Gly</td><td>Arg</td><td>Gly</td><td>Leu 500</td><td>Thr</td><td>Ala</td><td>Gly</td><td>Glh</td><td>Gin 505</td><td>Arg</td><td>Arg</td><td>Arg</td><td>Thr</td><td>Ile 510</td><td>Gly</td><td>Arg</td>

<td>Ser</td><td>Leu</td><td>Ala 515</td><td>Leu</td><td>Ser</td><td>Gly</td><td>Tyr</td><td>Val 520</td><td>Glu</td><td>Ile</td><td>Leu</td><td>Pro</td><td>Thr 525</td><td>Pro</td><td>Phe</td><td>Leu</td>

<td>Pro</td><td>Ala 53 0</td><td>Gly</td><td>val</td><td>Phe</td><td>Asp</td><td>Leu 535</td><td>Trp</td><td>Gly</td><td>Leu</td><td>Glu</td><td>Ala 540</td><td>Asp</td><td>Asp</td><td>Ser</td><td>Arg</td>

<td>Arg 545</td><td>Met</td><td>Thr</td><td>Thr</td><td>Arg</td><td>Val 550</td><td>Leu</td><td>Asn</td><td>Pro</td><td>Leu</td><td>Glu 555</td><td>Ala</td><td>Asp</td><td>Arg</td><td>pro</td><td>Gin 560</td>

<td>Leu</td><td>Ala</td><td>Thr</td><td>Thr</td><td>Leu 565</td><td>Leu</td><td>Pro</td><td>Ala</td><td>Leu</td><td>Leu 570</td><td>Glu</td><td>Ala</td><td>Leu</td><td>Val</td><td>Arg 575</td><td>Asn</td>

<td>Val</td><td>Ser</td><td>Arg</td><td>Gly 580</td><td>Leu</td><td>Val</td><td>Asp</td><td>Val</td><td>Ala 585</td><td>Leu</td><td>Phe</td><td>Ala</td><td>Ile</td><td>Ala 590</td><td>Gin</td><td>Val</td>

<td>Val</td><td>Gin</td><td>Pro 595</td><td>Thr</td><td>Glu</td><td>Gin</td><td>Thr</td><td>Arg 600</td><td>Gly</td><td>val</td><td>Gly</td><td>Leu</td><td>ile 605</td><td>Pro</td><td>Val</td><td>Asp</td>

<td>Arg</td><td>Arg 610</td><td>Pro</td><td>Thr</td><td>Asp</td><td>Asp</td><td>Glu 615</td><td>Ile</td><td>Ala</td><td>Met</td><td>Leu</td><td>Asp 620</td><td>Ala</td><td>Ser</td><td>Leu</td><td>pro</td>

<td>Arg 625</td><td>Gin</td><td>Pro</td><td>Gin</td><td>His</td><td>Val 630</td><td>Ala</td><td>Ala</td><td>Val</td><td>Leu</td><td>Ala 635</td><td>Gly</td><td>Leu</td><td>Arg</td><td>Glu</td><td>Pro 640</td>

<td>Arg</td><td>Gly</td><td>Pro</td><td>Trp</td><td>Gly 645</td><td>Pro</td><td>Gly</td><td>Arg</td><td>Pro</td><td>Val 650</td><td>Glu</td><td>Ala</td><td>Ala</td><td>Aap</td><td>Ala 655</td><td>Phe</td>

<td>Glu</td><td>Ala</td><td>Val</td><td>Arg 660</td><td>Ile</td><td>Ile</td><td>Ala</td><td>Arg</td><td>Ala 665</td><td>Ser</td><td>Arg</td><td>Val</td><td>Asp</td><td>Val 670</td><td>Thr</td><td>Leu</td>

<td>Arg</td><td>Pro</td><td>Ala 675</td><td>Gin</td><td>Tyr</td><td>Leu</td><td>Pro</td><td>Trp 680</td><td>His</td><td>Pro</td><td>Gly</td><td>Arg</td><td>Cys 685</td><td>Ala</td><td>Gin</td><td>Val</td>

<td>Phe</td><td>Val 690</td><td>Gly</td><td>Glu</td><td>Ser</td><td>Ser</td><td>Val 695</td><td>Gly</td><td>His</td><td>Ala</td><td>Gly</td><td>Gin 700</td><td>Leu</td><td>His</td><td>Pro</td><td>Ala</td>

<td>Val 705</td><td>Ile</td><td>Glu</td><td>Arg</td><td>Ser</td><td>Gly 710</td><td>Leu</td><td>Pro</td><td>Lys</td><td>Gly</td><td>Thr 715</td><td>Cys</td><td>Ala</td><td>val</td><td>Glu</td><td>Leu 720</td>

<td>Asn</td><td>Leu</td><td>Asp</td><td>Ala</td><td>Ile 725</td><td>Pro</td><td>Cys</td><td>Ser</td><td>Ala</td><td>Pro 730</td><td>Leu</td><td>Pro</td><td>Ala</td><td>Pro</td><td>Arg 735</td><td>Val</td>

<td>Ser</td><td>Pro</td><td>Tyr</td><td>Pro 740</td><td>Ala</td><td>Val</td><td>Phe</td><td>Gin</td><td>Asp 745</td><td>val</td><td>Ser</td><td>Leu</td><td>Val</td><td>Val 750</td><td>Ala</td><td>Ala</td>

<td>Aap</td><td>Ile</td><td>Pro⁷⁵⁵</td><td>Ala</td><td>Gin</td><td>Ala</td><td>Val</td><td>Ala 760</td><td>Asp</td><td>Ala</td><td>Val</td><td>Arg</td><td>Ala 765</td><td>Gly</td><td>Ala</td><td>Gly</td>

<td>Asp</td><td>Leu 770</td><td>Leu</td><td>Glu</td><td>Asp</td><td>Ile</td><td>Ala 775</td><td>Leu</td><td>Phe</td><td>Asp</td><td>Val</td><td>Phe 7B0</td><td>Thr</td><td>Gly</td><td>Pro</td><td>Gin</td>

<td>Ile 785</td><td>Gly</td><td>Glu</td><td>His</td><td>Arg</td><td>Lys 790</td><td>Ser</td><td>Leu</td><td>Thr</td><td>Phe</td><td>Ala 795</td><td>Leu</td><td>Arg</td><td>Phe</td><td>Arg</td><td>Ala 800</td>

<td>Pro</td><td>Asp</td><td>Arg</td><td>Thr</td><td>Leu 805</td><td>Thr</td><td>Glu</td><td>Asp</td><td>Asp</td><td>Ala 810</td><td>Ser</td><td>Ala</td><td>Ala</td><td>Arg</td><td>Asp 815</td><td>Ala</td>

<td>Ala</td><td>Val</td><td>Gin</td><td>Ser 820</td><td>Ala</td><td>Ala</td><td>Glu</td><td>Arg</td><td>Val 825</td><td>Gly</td><td>Ala</td><td>Val</td><td>Leu</td><td>Arg 330</td><td>Gly</td><td></td>

&lt;210&gt; 90 &lt;211&gt; 764 &lt;212&gt; PRT &lt;213&gt; Helicobacter pylori &lt;400&gt; 90

<td>Met 1</td><td>Lys</td><td>Leu</td><td>Ser</td><td>Ile 5</td><td>Asn</td><td>Asp</td><td>Leu</td><td>Asn</td><td>val 10</td><td>Phe</td><td>Val</td><td>Asn</td><td>Thr</td><td>Pro 15</td><td>Lys</td>

<td>Asp</td><td>Ile</td><td>Ala</td><td>Lys 20</td><td>Leu</td><td>Cys</td><td>Glu</td><td>Asp</td><td>Leu 25</td><td>Ser</td><td>Arg</td><td>Leu</td><td>Gly</td><td>Leu 30</td><td>Glu</td><td>Val</td>

<td>Glu</td><td>Ser</td><td>Cys</td><td>Ile</td><td>Pro</td><td>Cys</td><td>Ile</td><td>Ala</td><td>Pro</td><td>Lys</td><td>Asn</td><td>Val</td><td>Val</td><td>Val</td><td>Gly</td><td>Lys</td>

-165-

<td>Ile</td><td>Leu</td><td>35 Glu</td><td>Lys</td><td>Ala</td><td>Pro</td><td>His</td><td>40 Lye</td><td>Asn</td><td>Ala</td><td>GlU</td><td>Lys</td><td>45 Leu</td><td>Ser</td><td>Val</td><td>Cys</td>

<td>Gin</td><td>50 Val</td><td>Asp</td><td>Val</td><td>Gly</td><td>Lys</td><td>55 Glu</td><td>Val</td><td>Leu</td><td>Gin</td><td>Ile</td><td>60 Val</td><td>Cys</td><td>Gly</td><td>Ala</td><td>Lys</td>

<td>65 Asn</td><td>Val</td><td>Ala</td><td>Pro</td><td>Asn</td><td>70 Gin</td><td>Phe</td><td>Val</td><td>Pro</td><td>Val</td><td>75 Ala</td><td>Leu</td><td>Asn</td><td>Gly</td><td>Ala</td><td>80 Leu</td>

<td>Ile</td><td>Gly</td><td>Ser</td><td>Thr</td><td>85 Thr</td><td>Ile</td><td>Ala</td><td>Lys</td><td>Thr</td><td>90 Glu</td><td>Leu</td><td>Arg</td><td>Gly</td><td>Val</td><td>95 Glu</td><td>Ser</td>

<td>His</td><td>Gly</td><td>Met</td><td>100 Ile</td><td>Cys</td><td>Ser</td><td>Ser</td><td>Ile</td><td>105 Glu</td><td>Leu</td><td>Gly</td><td>Phe</td><td>Pro</td><td>110 Lys</td><td>Ile</td><td>Asn</td>

<td>Asp</td><td>Gly</td><td>115 Ile</td><td>Leu</td><td>Glu</td><td>Leu</td><td>Asp</td><td>120 Glu</td><td>Ser</td><td>Val</td><td>Gly</td><td>Glu</td><td>125 Leu</td><td>Val</td><td>Leu</td><td>Gly</td>

<td>Lys</td><td>130 Glu</td><td>Leu</td><td>Asn</td><td>Glu</td><td>Tyr</td><td>135 Ala</td><td>Pro</td><td>Phe</td><td>Asn</td><td>Thr</td><td>140 His</td><td>V*1</td><td>Leu</td><td>Glu</td><td>Ile</td>

<td>14 5 Ser</td><td>Leu</td><td>Thr</td><td>Pro</td><td>Asn</td><td>150 Arg</td><td>Gly</td><td>Asp</td><td>Cys</td><td>Leu</td><td>155 Ser</td><td>Val</td><td>Leu</td><td>Gly</td><td>Ile</td><td>160 Ala</td>

<td>Arg</td><td>Glu</td><td>Ile</td><td>Ser</td><td>165 Ala</td><td>Phe</td><td>Tyr</td><td>His</td><td>Thr</td><td>170 Pro</td><td>Leu</td><td>Lys</td><td>pro</td><td>ile</td><td>175 Lys</td><td>Ala</td>

<td>Leu</td><td>Asn</td><td>Phe</td><td>180 Thr</td><td>Pro</td><td>Lys</td><td>Ser</td><td>Gly</td><td>185 Leu</td><td>Ile</td><td>Thr</td><td>Leu</td><td>Ser</td><td>190 Ala</td><td>Gly</td><td>Glu</td>

<td>Asn</td><td>Ile</td><td>195 Glu</td><td>Ser</td><td>His</td><td>Leu</td><td>Ala</td><td>200 Tyr</td><td>Tyr</td><td>Leu</td><td>Ile</td><td>Cys</td><td>205 Asn</td><td>His</td><td>Ser</td><td>Leu</td>

<td>Lys</td><td>210 Thr</td><td>Pro</td><td>Leu</td><td>Asn</td><td>Ile</td><td>215 Lys</td><td>Leu</td><td>Ser</td><td>Leu</td><td>Ala</td><td>220 His</td><td>Asn</td><td>Asn</td><td>Ala</td><td>Leu</td>

<td>225 Ser</td><td>Glu</td><td>Asn</td><td>Asp</td><td>Leu</td><td>230 Asn</td><td>Asn</td><td>Phe</td><td>Ile</td><td>Glu</td><td>235 Phe</td><td>Ser</td><td>Thr</td><td>His</td><td>Phe</td><td>240 Ser</td>

<td>Gly</td><td>Val</td><td>Ile</td><td>Met</td><td>245 Asn</td><td>Ala</td><td>Tyr</td><td>Ser</td><td>Leu</td><td>250 Asn</td><td>Thr</td><td>Thr</td><td>Pro</td><td>Met</td><td>255 Asp</td><td>Leu</td>

<td>Ser</td><td>Val</td><td>Lys</td><td>260 Asn</td><td>Asp</td><td>Glu</td><td>Asn</td><td>Asn</td><td>265 Leu</td><td>Glu</td><td>Ser</td><td>Val</td><td>Tyr</td><td>270 Ile</td><td>Asn</td><td>His</td>

<td>Gin</td><td>Lys</td><td>275 Arg</td><td>Ser</td><td>Thr</td><td>Ile</td><td>Ala</td><td>280 Ile</td><td>Lys</td><td>His</td><td>Gin</td><td>Val</td><td>285 Gin</td><td>Lyś</td><td>Asp</td><td>Leu</td>

<td>Ser</td><td>290 Glu</td><td>Cys</td><td>Leu</td><td>Leu</td><td>Leu</td><td>295 Glu</td><td>Ala</td><td>Ser</td><td>Tyr</td><td>Thr</td><td>300 Asp</td><td>pro</td><td>Ile</td><td>Ser</td><td>Leu</td>

<td>305 Ser</td><td>Leu</td><td>Lys</td><td>Leu</td><td>Kis</td><td>310 Ala</td><td>Leu</td><td>Lys</td><td>Asp</td><td>Lys</td><td>315 Thr</td><td>Leu</td><td>Gin</td><td>Lys</td><td>Asp</td><td>320 Asn</td>

<td>Ala</td><td>Leu</td><td>Ile</td><td>Tyr</td><td>325 Arg</td><td>Ser</td><td>Ala</td><td>Arg</td><td>Gly</td><td>330 Ser</td><td>Asn</td><td>Pro</td><td>Asn</td><td>Leu</td><td>335 Ser</td><td>Asp</td>

<td>Gly</td><td>Leu</td><td>Asn</td><td>340 Phe</td><td>Leu</td><td>Ser</td><td>Ala</td><td>His</td><td>345 Leu</td><td>Lys</td><td>Ala</td><td>Thr</td><td>Ile</td><td>350 Leu</td><td>Glu</td><td>Ser</td>

<td>Lys</td><td>Gin</td><td>355 Thr</td><td>Glu</td><td>His</td><td>Ser</td><td>Leu</td><td>360 Lys</td><td>Asp</td><td>Arg</td><td>Thr</td><td>Leu</td><td>365 Thr</td><td>Phe</td><td>Gin</td><td>Leu</td>

<td>Glu</td><td>370 Asp</td><td>Ile</td><td>Thr</td><td>Glu</td><td>Ile</td><td>375 Leu</td><td>Gly</td><td>Leu</td><td>Ala</td><td>Val</td><td>380 Glu</td><td>Lys</td><td>Glu</td><td>Lys</td><td>Ile</td>

<td>385 Gin</td><td>Gly</td><td>Ile</td><td>Leu</td><td>Lys</td><td>390 Asn</td><td>Leu</td><td>Gly</td><td>Phe</td><td>Lys</td><td>395 Val</td><td>Ser</td><td>Val</td><td>Lys</td><td>Glu</td><td>400 Pro</td>

<td>Asn</td><td>Ser</td><td>Lys</td><td>Pro</td><td>405 Gin</td><td>Ile</td><td>Leu</td><td>Glu</td><td>Val</td><td>410 Ile</td><td>Ala</td><td>Pro</td><td>Asn</td><td>Phe</td><td>415 Arg</td><td>His</td>

<td>Asp</td><td>Ile</td><td>Lys</td><td>420 Thr</td><td>Ile</td><td>Gin</td><td>Asp</td><td>Ile</td><td>425 Ala</td><td>Glu</td><td>Glu</td><td>Ile</td><td>Leu</td><td>430 Arg</td><td>Phe</td><td>Val</td>

<td>Gly</td><td>Ile</td><td>435 Asp</td><td>Asn</td><td>Leu</td><td>Val</td><td>Ser</td><td>440 Lys</td><td>pro</td><td>Leu</td><td>His</td><td>Cys</td><td>445 Val</td><td>ser</td><td>Ser</td><td>Lys</td>

<td>Asn</td><td>450 Ser</td><td>Asn</td><td>Pro</td><td>Asn</td><td>Tyr</td><td>455 Asp</td><td>Thr</td><td>His</td><td>Arg</td><td>Phe</td><td>460 Phe</td><td>Glu</td><td>Asn</td><td>Leu</td><td>Lys</td>

<td>465 His</td><td>Lys</td><td>Ala</td><td>Leu</td><td>Ala</td><td>470 Cys</td><td>Gly</td><td>Phe</td><td>Lys</td><td>Glu</td><td>475 Val</td><td>Ile</td><td>His</td><td>Tyr</td><td>Val</td><td>480 Phe</td>

<td>Tyr</td><td>Ser</td><td>Lys</td><td>Glu</td><td>485 Lys</td><td>Gin</td><td>Gin</td><td>Lys</td><td>Leu</td><td>490 Gly</td><td>Phe</td><td>Glu</td><td>Val</td><td>Leu</td><td>495 Glu</td><td>Asp</td>

-166-

<td>Pro</td><td>Leu</td><td>Glu</td><td>500 Leu</td><td>Gln</td><td>Aśn</td><td>Pro</td><td>Ile</td><td>505 Thr</td><td>Thr</td><td>Glu</td><td>Leu</td><td>Asn</td><td>510 Thr</td><td>Leu</td><td>Arg</td>

<td>Thr</td><td>Ser</td><td>515 Leu</td><td>Val</td><td>Cys</td><td>Gly</td><td>Leu</td><td>520 Leu</td><td>Asp</td><td>Ala</td><td>Ser</td><td>Leu</td><td>525 Arg</td><td>Asn</td><td>Lys</td><td>Asn</td>

<td>Leu</td><td>530 Gly</td><td>Phe</td><td>Lys</td><td>Ser</td><td>Ile</td><td>535 Ala</td><td>Leu</td><td>Tyr</td><td>Glu</td><td>Lys</td><td>540 Gly</td><td>Ser</td><td>Val</td><td>Tyr</td><td>Asn</td>

<td>545 Ser</td><td>Lys</td><td>Arg</td><td>Glu</td><td>Glu</td><td>550 He</td><td>Gln</td><td>Lys</td><td>Leu</td><td>Gly</td><td>555 Phe</td><td>Leu</td><td>Ile</td><td>Ser</td><td>Gly</td><td>560 Leu</td>

<td>Gln</td><td>Lys</td><td>Lys</td><td>Glu</td><td>565 Ser</td><td>Tyr</td><td>Pro</td><td>Asp</td><td>Thr</td><td>570 Ly3</td><td>Gly</td><td>Lys</td><td>Ala</td><td>Trp</td><td>575 Asp</td><td>Phe</td>

<td>Tyr</td><td>Ser</td><td>Phe</td><td>580 Ala</td><td>Glu</td><td>Cys</td><td>Val</td><td>Ser</td><td>585 Lys</td><td>Val</td><td>Ile</td><td>Gly</td><td>Asp</td><td>590 Phe</td><td>Ser</td><td>Leu</td>

<td>Glu</td><td>Lys</td><td>595 Leu</td><td>Thr</td><td>Thr</td><td>Gln</td><td>Thr</td><td>600 Pro</td><td>Ile</td><td>Asn</td><td>His</td><td>Pro</td><td>605 Tyr</td><td>Gln</td><td>Ser</td><td>Ala</td>

<td>Lya</td><td>610 Ile</td><td>ile</td><td>Gln</td><td>Asn</td><td>His</td><td>615 Glu</td><td>Ile</td><td>Ile</td><td>Gly</td><td>val</td><td>620 Ile</td><td>Ala</td><td>Lys</td><td>Ile</td><td>His</td>

<td>625 Pro</td><td>Lys</td><td>Val</td><td>Ile</td><td>Gln</td><td>630 Glu</td><td>Leu</td><td>Asp</td><td>Leu</td><td>Phe</td><td>635 Glu</td><td>Ser</td><td>Tyr</td><td>Tyr</td><td>Ala</td><td>640 GlU</td>

<td>Ile</td><td>Asp</td><td>Ala</td><td>Phe</td><td>645 Lys</td><td>Leu</td><td>Lys</td><td>Arg</td><td>Pro</td><td>650 Ala</td><td>Met</td><td>Leu</td><td>Leu</td><td>Lys</td><td>655 Pro</td><td>Phe</td>

<td>Ser</td><td>Ile</td><td>Tyr</td><td>660 Pro</td><td>Ser</td><td>Ser</td><td>Val</td><td>Arg</td><td>665 Asp</td><td>Leu</td><td>Thr</td><td>Leu</td><td>Ile</td><td>670 Ile</td><td>Asp</td><td>Glu</td>

<td>Asn</td><td>Thr</td><td>675 Ala</td><td>Phe</td><td>Ser</td><td>Gly</td><td>Ile</td><td>680 Lys</td><td>Lys</td><td>Ala</td><td>Leu</td><td>Lys</td><td>685 Asp</td><td>Ala</td><td>Gln</td><td>Ile</td>

<td>Pro</td><td>690 Asn</td><td>Leu</td><td>ser</td><td>Glu</td><td>Ile</td><td>695 Leu</td><td>Pro</td><td>Leu</td><td>Asp</td><td>Ile</td><td>700 Phe</td><td>Lys</td><td>Glu</td><td>Ser</td><td>Asn</td>

<td>705 Asn</td><td>Ser</td><td>Ile</td><td>Ala</td><td>Leu</td><td>710 Ser</td><td>Val</td><td>Arg</td><td>Cys</td><td>Val</td><td>715 Ile</td><td>His</td><td>Ser</td><td>Leu</td><td>Glu</td><td>720 Lys</td>

<td>Thr</td><td>Leu</td><td>Asn</td><td>Asp</td><td>725 Glu</td><td>Glu</td><td>Val</td><td>Asn</td><td>Ser</td><td>730 Ala</td><td>Val</td><td>Gln</td><td>Lys</td><td>Ala</td><td>735 Leu</td><td>Glu</td>

<td>Ile</td><td>Leu</td><td>Glu 755</td><td>740 Lys</td><td>GlU</td><td>Phe</td><td>Asn</td><td>Ala 760</td><td>745 Arg</td><td>Leu</td><td>Lys</td><td>Gly</td><td></td><td> 750</td><td></td><td></td>

&lt;210&gt; 91 &lt;211&gt; 792 &lt;212&gt; PRT &lt;213&gt; Pseudomonas aeruginosa &lt;400&gt; 91

<td>Met 1</td><td>Lys</td><td>phe</td><td>Ser</td><td>Glu 5</td><td>Lys</td><td>Trp</td><td>Leu</td><td>Arg</td><td>Ser 10</td><td>Trp</td><td>Ala</td><td>Asn</td><td>Pro</td><td>Gln 15</td><td>Val</td>

<td>Ser</td><td>His</td><td>Asp</td><td>Glu 20</td><td>Leu</td><td>Val</td><td>Ala</td><td>Arg</td><td>Leu 25</td><td>Ser</td><td>Met</td><td>Val</td><td>Gly</td><td>Leu 30</td><td>Glu</td><td>Val</td>

<td>Asp</td><td>Ala</td><td>Asp 35</td><td>Leu</td><td>Pro</td><td>val</td><td>Ala</td><td>Gly 40</td><td>Ala</td><td>Phe</td><td>Ser</td><td>Gly</td><td>Val 45</td><td>Val</td><td>val</td><td>Gly</td>

<td>GlU</td><td>Val 50</td><td>Leu</td><td>Ser</td><td>Thr</td><td>Glu</td><td>Gln 55</td><td>His</td><td>Pro</td><td>Asp</td><td>Ala</td><td>Asp 60</td><td>Lys</td><td>Leu</td><td>Arg</td><td>Val</td>

<td>Cys 65</td><td>Gln</td><td>val</td><td>Ser</td><td>Asn</td><td>Gly 70</td><td>Ser</td><td>Glu</td><td>Thr</td><td>Phe</td><td>Gln 75</td><td>val</td><td>Val</td><td>cys</td><td>Gly</td><td>Ala 80</td>

<td>Pro</td><td>Asn</td><td>Val</td><td>Arg</td><td>Ala 85</td><td>Gly</td><td>Leu</td><td>Lys</td><td>He</td><td>Pro 90</td><td>Phe</td><td>Ala</td><td>Met</td><td>Ile</td><td>Gly 95</td><td>Ala</td>

<td>Glu</td><td>Leu</td><td>Pro</td><td>Asp 100</td><td>Asp</td><td>Phe</td><td>Lys</td><td>Ile</td><td>Lys 105</td><td>Lys</td><td>Ala</td><td>Lys</td><td>Leu</td><td>Arg 110</td><td>Gly</td><td>val</td>

<td>Glu</td><td>Ser</td><td>Phe 115</td><td>Gly</td><td>Met</td><td>Leu</td><td>Cys</td><td>Ser 120</td><td>Ala</td><td>Lys</td><td>Glu</td><td>Leu</td><td>Gln 125</td><td>Ile</td><td>Ser</td><td>Glu</td>

<td>Glu</td><td>Asn</td><td>Ala</td><td>Gly</td><td>Leu</td><td>Leu</td><td>Glu</td><td>Leu</td><td>Pro</td><td>Ala</td><td>Asp</td><td>Ala</td><td>Pro</td><td>Val</td><td>Gly</td><td>Gln</td>

-167-

<td>Asp</td><td>130 Val</td><td>Arg</td><td>Thr</td><td>Tyr</td><td>Leu</td><td>135 Glu</td><td>Leu</td><td>Ala</td><td>ASP</td><td>Tyr</td><td>14 0 Thr</td><td>Ile</td><td>Glu</td><td>Val</td><td>Gly</td>

<td>145 Leu</td><td>Thr</td><td>Pro</td><td>Asn</td><td>Arg</td><td>150 Gly</td><td>Asp</td><td>Cys</td><td>Leu</td><td>Ser</td><td>155 Leu</td><td>Ala</td><td>Gly</td><td>Leu</td><td>Ala</td><td>160 Arg</td>

<td>Glu</td><td>Val</td><td>Ser</td><td>Ala</td><td>165 Ile</td><td>Tyr</td><td>Asp</td><td>Val</td><td>Pro</td><td>170 Leu</td><td>Ala</td><td>Pro</td><td>Val</td><td>Ala</td><td>175 val</td><td>Asp</td>

<td>Ala</td><td>Val</td><td>Ala</td><td>180 Ala</td><td>Gin</td><td>His</td><td>ASp</td><td>Glu</td><td>135 Thr</td><td>Arg</td><td>Pro</td><td>Val</td><td>Glu</td><td>190 Leu</td><td>Ala</td><td>Ala</td>

<td>Pro</td><td>Ala</td><td>195 Ala</td><td>Cys</td><td>Pro</td><td>Arg</td><td>Tyr</td><td>200 Leu</td><td>Gly</td><td>Arg</td><td>val</td><td>Ile</td><td>205 Arg</td><td>Asn</td><td>Val</td><td>Asp</td>

<td>Leu</td><td>210 Ser</td><td>Arg</td><td>Pro</td><td>Thr</td><td>Pro</td><td>215 Leu</td><td>Trp</td><td>Met</td><td>Val</td><td>Glu</td><td>220 Arg</td><td>Leu</td><td>Arg</td><td>Arg</td><td>Ser</td>

<td>225 Asp</td><td>Ile</td><td>Arg</td><td>Ser</td><td>Ile</td><td>230 Asp</td><td>pro</td><td>Val</td><td>Val</td><td>Asp</td><td>235 Val</td><td>Thr</td><td>Asn</td><td>Tyr</td><td>Val</td><td>240 Met</td>

<td>Ile</td><td>Glu</td><td>Leu</td><td>Gly</td><td>245 Gin</td><td>Pro</td><td>Met</td><td>His</td><td>Ala</td><td>250 Phe</td><td>Asp</td><td>Leu</td><td>Ala</td><td>Glu</td><td>255 Ile</td><td>Asn</td>

<td>Gly</td><td>Gly</td><td>Val</td><td>260 Arg</td><td>Val</td><td>Arg</td><td>Met</td><td>Ala</td><td>265 Glu</td><td>Asp</td><td>Gly</td><td>Glu</td><td>Lys</td><td>270 Leu</td><td>Val</td><td>Leu</td>

<td>Leu</td><td>Asp</td><td>275 Gly</td><td>Gin</td><td>Glu</td><td>Ile</td><td>Thr</td><td>280 Leu</td><td>Arg</td><td>Ala</td><td>Asp</td><td>Thr</td><td>285 Leu</td><td>Val</td><td>Ile</td><td>Ala</td>

<td>Asp</td><td>290 His</td><td>Gin</td><td>Arg</td><td>Ala</td><td>Leu</td><td>295 Ala</td><td>ile</td><td>Ala</td><td>Gly</td><td>Val</td><td>300 Met</td><td>Gly</td><td>Gly</td><td>Glu</td><td>His</td>

<td>305 Ser</td><td>Gly</td><td>Val</td><td>Ser</td><td>Asp</td><td>310 Ser</td><td>Thr</td><td>Arg</td><td>Asp</td><td>Leu</td><td>315 Phe</td><td>Leu</td><td>Glu</td><td>Ala</td><td>Ala</td><td>320 Phe</td>

<td>Phe</td><td>Asp</td><td>Thr</td><td>Ile</td><td>325 Ala</td><td>Leu</td><td>Ala</td><td>Gly</td><td>Lys</td><td>330 Ala</td><td>Arg</td><td>Ser</td><td>Tyr</td><td>Gly</td><td>335 Leu</td><td>His</td>

<td>Thr</td><td>Asp</td><td>Ser</td><td>340 Ser</td><td>His</td><td>Arg</td><td>Phe</td><td>Glu</td><td>345 Arg</td><td>Gly</td><td>val</td><td>Asp</td><td>Ser</td><td>350' Gin</td><td>Leu</td><td>Ala</td>

<td>Arg</td><td>Lys</td><td>355 Ala</td><td>Met</td><td>Glu</td><td>Arg</td><td>Ala</td><td>360 Thr</td><td>Arg</td><td>Leu</td><td>Ile</td><td>Leu</td><td>365 Asp</td><td>Ile</td><td>Val</td><td>Gly</td>

<td>Gly</td><td>370 Glu</td><td>Pro</td><td>Gly</td><td>Pro</td><td>Ile</td><td>375 Val</td><td>Glu</td><td>Gin</td><td>Val</td><td>Ser</td><td>380 Glu</td><td>Ala</td><td>His</td><td>Leu</td><td>Pro</td>

<td>385 Lys</td><td>Val</td><td>Ala</td><td>Pro</td><td>Ile</td><td>390 Thr</td><td>Leu</td><td>Arg</td><td>Ala</td><td>Glu</td><td>395 Arg</td><td>Val</td><td>Thr</td><td>Gin</td><td>Met</td><td>400 Leu</td>

<td>Gly</td><td>Met</td><td>Pro</td><td>Leu</td><td>405 Asp</td><td>Ala</td><td>Ala</td><td>Glu</td><td>Ile</td><td>410 val</td><td>Arg</td><td>Leu</td><td>Leu</td><td>Gin</td><td>415 Ala</td><td>Leu</td>

<td>Glu</td><td>Leu</td><td>Thr</td><td>420 Val</td><td>Val</td><td>Ala</td><td>Asp</td><td>Gly</td><td>425 Glu</td><td>Gly</td><td>Gin</td><td>Trp</td><td>Ser</td><td>430 Val</td><td>Gly</td><td>val</td>

<td>Pro</td><td>Ser</td><td>435 His</td><td>Arg</td><td>Phe</td><td>Asp</td><td>Ile</td><td>440 Ser</td><td>Leu</td><td>Glu</td><td>Val</td><td>Asp</td><td>445 Leu</td><td>Ile</td><td>Glu</td><td>Glu</td>

<td>Leu</td><td>450 Ala</td><td>Arg</td><td>Leu</td><td>Tyr</td><td>Gly</td><td>455 Tyr</td><td>Asn</td><td>Arg</td><td>Leu</td><td>pro</td><td>460 Val</td><td>Arg</td><td>Tyr</td><td>Pro</td><td>Gin</td>

<td>465 Ala</td><td>Arg</td><td>Leu</td><td>Ala</td><td>Pro</td><td>470 Asn</td><td>Asn</td><td>Lys</td><td>pro</td><td>Glu</td><td>475 Ala</td><td>Arg</td><td>Ala</td><td>Ala</td><td>Leu</td><td>480 Pro</td>

<td>Leu</td><td>Leu</td><td>Arg</td><td>Arg</td><td>485 Leu</td><td>Leu</td><td>vai</td><td>Ala</td><td>Arg</td><td>490 Gly</td><td>Tyr</td><td>Gin</td><td>Glu</td><td>Ala</td><td>495 Ile</td><td>Thr</td>

<td>Phe</td><td>Ser</td><td>Phe</td><td>500 Ile</td><td>Asp</td><td>Pro</td><td>Ala</td><td>Leu</td><td>505 Phe</td><td>Glu</td><td>Leu</td><td>Phe</td><td>Asp</td><td>510 Pro</td><td>Gly</td><td>Thr</td>

<td>Gin</td><td>Pro</td><td>SIS Leu</td><td>Thr</td><td>Leu</td><td>Ala</td><td>Asn</td><td>520 Pro</td><td>Ile</td><td>Ser</td><td>Ala</td><td>Asp</td><td>S25 Met</td><td>Ala</td><td>Ala</td><td>Met</td>

<td>Arg</td><td>530 Ser</td><td>Ser</td><td>Leu</td><td>Trp</td><td>Pro</td><td>535 Gly</td><td>Leu</td><td>Val</td><td>Lys</td><td>Ala</td><td>540 Leu</td><td>Gin</td><td>His</td><td>Asn</td><td>Leu</td>

<td>545 •Asn</td><td>Arg</td><td>Gin</td><td>Gin</td><td>Ser</td><td>550 Arg</td><td>Val</td><td>Arg</td><td>Leu</td><td>Phe</td><td>555 Glu</td><td>Ser</td><td>Gly</td><td>Leu</td><td>Arg</td><td>560 Phe</td>

<td>Val</td><td>Gly</td><td>Gin</td><td>Leu</td><td>565 Glu</td><td>Gly</td><td>Leu</td><td>Lys</td><td>Gin</td><td>570 Glu</td><td>Ala</td><td>Met</td><td>Leu</td><td>Ala</td><td>575 Gly</td><td>Ala</td>

<td>Ile</td><td>Cys</td><td>Gly</td><td>580 Lys</td><td>Arg</td><td>Leu</td><td>Pro</td><td>Glu</td><td>585 Gly</td><td>Trp</td><td>Ala</td><td>Asn</td><td>Gly</td><td>590 Arg</td><td>Asp</td><td>Gly</td>

-168595 600 605

<td rowspan="2">Val</td><td rowspan="2">Asp 610</td><td rowspan="2">Phe</td><td rowspan="2">Phe</td><td colspan="2" rowspan="2">Asp Ala</td><td colspan="9">Lys Ala Asp Val Glu Ala Val Leu Ala</td><td rowspan="2">Ser</td>

<td colspan="4"> 615</td><td colspan="5"> 620</td>

<td>Ala</td><td>Gly</td><td>Ala</td><td>Leu</td><td>Gly</td><td>Asp</td><td>Phe</td><td>Ser</td><td>Phe</td><td>Val</td><td>Pro</td><td>Gly</td><td>Glu</td><td>His</td><td>Pro</td><td>Ala</td>

<td> 625</td><td></td><td></td><td></td><td></td><td> 630</td><td></td><td></td><td></td><td></td><td> 635</td><td></td><td></td><td></td><td></td><td> 640</td>

<td>Leu</td><td>His</td><td>Pro</td><td>Gly</td><td>Gin</td><td>Thr</td><td>Ala</td><td>Arg</td><td>Ile</td><td>Glu</td><td>Arg</td><td>Glu</td><td>Gly</td><td>Arg</td><td>Leu</td><td>Val</td>

<td></td><td></td><td></td><td></td><td> 645</td><td></td><td></td><td></td><td></td><td> 650</td><td></td><td></td><td></td><td></td><td> 655</td><td></td>

<td>Gly</td><td>Tyr</td><td>Leu</td><td>Gly</td><td>Ala</td><td>Leu</td><td>His</td><td>Pro</td><td>Glu</td><td>Leu</td><td>Ala</td><td>Lys</td><td>Ly3</td><td>Leu</td><td>Asp</td><td>Leu</td>

<td></td><td></td><td></td><td> 660</td><td></td><td></td><td></td><td></td><td> 665</td><td></td><td></td><td></td><td></td><td> 670</td><td></td><td></td>

<td>Glu</td><td>Gin</td><td>Pro</td><td>Val</td><td>Phe</td><td>Leu</td><td>Phe</td><td>Glu</td><td>Leu</td><td>Leu</td><td>Leu</td><td>Ala</td><td>Glu</td><td>Val</td><td>Val</td><td>Asp</td>

<td></td><td></td><td> 675</td><td></td><td></td><td></td><td></td><td> 680</td><td></td><td></td><td></td><td></td><td> 685</td><td></td><td></td><td></td>

<td>Gly</td><td>His</td><td>Leu</td><td>Pro</td><td>Lys</td><td>Phe</td><td>Arg</td><td>Glu</td><td>Leu</td><td>Ser</td><td>Arg</td><td>Phe</td><td>Pro</td><td>Glu</td><td>Val</td><td>Arg</td>

<td></td><td> 690</td><td></td><td></td><td></td><td></td><td> 695</td><td></td><td></td><td></td><td></td><td> 700</td><td></td><td></td><td></td><td></td>

<td>Arg</td><td>Asp</td><td>Leu</td><td>Ala</td><td>Leu</td><td>Leu</td><td>Val</td><td>Asp</td><td>Gin</td><td>Asp</td><td>Val</td><td>Pro</td><td>Ala</td><td>Gin</td><td>Asp</td><td>Ile</td>

<td> 705</td><td></td><td></td><td></td><td></td><td> 710</td><td></td><td></td><td></td><td></td><td> 715</td><td></td><td></td><td></td><td></td><td> 720</td>

<td>Leu</td><td>Thr</td><td>Gin</td><td>Ile</td><td>Arg</td><td>Ala</td><td>Ala</td><td>Ala</td><td>Gly</td><td>Glu</td><td>Trp</td><td>Leu</td><td>Thr</td><td>Asp</td><td>Leu</td><td>Arg</td>

<td></td><td></td><td></td><td></td><td> 725</td><td></td><td></td><td></td><td></td><td> 730</td><td></td><td></td><td></td><td></td><td> 735</td><td></td>

<td>Leu</td><td>Phe</td><td>Aap</td><td>Val</td><td>Tyr</td><td>His</td><td>Gly</td><td>Lys</td><td>Gly</td><td>Ile</td><td>Asp</td><td>Pro</td><td>His</td><td>Arg</td><td>Lya</td><td>Ser</td>

<td></td><td></td><td></td><td> 740</td><td></td><td></td><td></td><td></td><td> 745</td><td></td><td></td><td></td><td></td><td> 750</td><td></td><td></td>

<td>Leu</td><td>Ala</td><td>Val</td><td>Gly</td><td>Leu</td><td>Thr</td><td>Trp</td><td>Gin</td><td>His</td><td>Pro</td><td>Ser</td><td>Arg</td><td>Thr</td><td>Leu</td><td>Asn</td><td>Asp</td>

<td></td><td></td><td> 755</td><td></td><td></td><td></td><td></td><td> 760</td><td></td><td></td><td></td><td></td><td> 765</td><td></td><td></td><td></td>

<td>Asp</td><td>Glu</td><td>Val</td><td>Asn</td><td>Ser</td><td>Thr</td><td>Thr</td><td>Gin</td><td>Aan</td><td>Ile</td><td>Val</td><td>Thr</td><td>Ser</td><td>Leu</td><td>Glu</td><td>Glu</td>

<td></td><td> 770</td><td></td><td></td><td></td><td></td><td> 775</td><td></td><td></td><td></td><td></td><td> 780</td><td></td><td></td><td></td><td></td>

<td>Arg</td><td>Phe</td><td>Asn</td><td>Ala</td><td>Thr</td><td>Leu</td><td>Arg</td><td>Lys</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

785 790 &lt;210&gt; 92 &lt;211&gt; 551 &lt;212&gt; PRT &lt;213&gt; Escherichia coli &lt;400&gt; 92

<td>Met 1</td><td>Thr</td><td>Gin</td><td>Val</td><td>Ala s</td><td>Lys</td><td>Lys</td><td>Ile</td><td>Leu</td><td>Val 10</td><td>Thr</td><td>Cys</td><td>Ala</td><td>Leu</td><td>Pro 15</td><td>Tyr</td>

<td>Ala</td><td>Asn</td><td>Gly</td><td>Ser 20</td><td>ile</td><td>His</td><td>Leu</td><td>Gly</td><td>His 25</td><td>Met</td><td>Leu</td><td>Glu</td><td>His</td><td>Ile 30</td><td>Gin</td><td>Ala</td>

<td>Asp</td><td>Val</td><td>Trp 35</td><td>Val</td><td>Arg</td><td>Tyr</td><td>Gin</td><td>Arg 40</td><td>Met</td><td>Arg</td><td>Gly</td><td>His</td><td>Glu 45</td><td>Val</td><td>Asn</td><td>Phe</td>

<td>Ile</td><td>Cys 50</td><td>Ala</td><td>Asp</td><td>ASP</td><td>Ala</td><td>His 55</td><td>Gly</td><td>Thr</td><td>Pro</td><td>Ile</td><td>Met 60</td><td>Leu</td><td>Lys</td><td>Ala</td><td>Gin</td>

<td>Gin 65</td><td>Leu</td><td>Gly</td><td>Ile</td><td>Thr</td><td>Pro 70</td><td>Glu</td><td>Gin</td><td>Met</td><td>Ile</td><td>Gly 75</td><td>Glu</td><td>Met</td><td>Ser</td><td>Gin</td><td>Glu 80</td>

<td>His</td><td>Gin</td><td>Thr</td><td>Asp</td><td>Phe 85</td><td>Ala</td><td>Gly</td><td>Phe</td><td>Asn</td><td>Ile 90</td><td>Ser</td><td>Tyr</td><td>Asp</td><td>Asn</td><td>Tyr 95</td><td>His</td>

<td>Ser</td><td>Thr</td><td>His</td><td>Ser 100</td><td>Glu</td><td>Glu</td><td>Asn</td><td>Arg</td><td>Gin 105</td><td>Leu</td><td>Ser</td><td>Glu</td><td>Leu</td><td>Ile 110</td><td>Tyr</td><td>Ser</td>

<td>Arg</td><td>Leu</td><td>Lys 115</td><td>Glu</td><td>Asn</td><td>Gly</td><td>Phe</td><td>Ile 120</td><td>Lys</td><td>Asn</td><td>Arg</td><td>Thr</td><td>Ile 125</td><td>Ser</td><td>Gin</td><td>Leu</td>

<td>Tyr</td><td>Asp 130</td><td>Pro</td><td>Glu</td><td>Lys</td><td>Gly</td><td>Met 135</td><td>Phe</td><td>Leu</td><td>Pro</td><td>Asp</td><td>Arg 140</td><td>Phe</td><td>Val</td><td>Lys</td><td>Gly</td>

<td>Thr 145</td><td>Cys</td><td>Pro</td><td>Lys</td><td>cys</td><td>Lys 150</td><td>Ser</td><td>pro</td><td>Asp</td><td>Gin</td><td>Tyr 155</td><td>Gly</td><td>Asp</td><td>Asn</td><td>Cys</td><td>Glu 160</td>

<td>val</td><td>Cys</td><td>Gly</td><td>Ala</td><td>Thr 165</td><td>Tyr</td><td>Ser</td><td>Pro</td><td>Thr</td><td>Glu 170</td><td>Leu</td><td>Ile</td><td>Glu</td><td>Pro</td><td>Lys 17S</td><td>Ser</td>

<td>Val</td><td>Val</td><td>Ser</td><td>Gly 180</td><td>Ala</td><td>Thr</td><td>Pro</td><td>val</td><td>Met 185</td><td>Arg</td><td>Asp</td><td>ser</td><td>Glu</td><td>His 190</td><td>phe</td><td>phe</td>

<td>Phe</td><td>Asp</td><td>Leu</td><td>Pro</td><td>Ser</td><td>Phe</td><td>Ser</td><td>Glu</td><td>Met</td><td>Leu</td><td>Gin</td><td>Ala</td><td>Trp</td><td>Thr</td><td>Arg</td><td>Ser</td>

-169-

<td>Gly</td><td>Ala</td><td>195 Leu</td><td>Gin</td><td>Glu</td><td>Gin</td><td>Val</td><td>200 Ala</td><td>Asn</td><td>Lys</td><td>Met</td><td>Gin</td><td>205 Glu</td><td>Trp</td><td>Phe</td><td>Glu</td>

<td>Ser</td><td>210 Gly</td><td>Leu</td><td>Gin</td><td>Gin</td><td>Trp</td><td>215 Asp</td><td>ile</td><td>Ser</td><td>Arg</td><td>Asp</td><td>220 Ala</td><td>Pro</td><td>Tyr</td><td>Phe</td><td>Gly</td>

<td>225 Phe</td><td>Glu</td><td>Ile</td><td>Pro</td><td>Asn</td><td>230 Ala</td><td>Pro</td><td>Gly</td><td>Lys</td><td>Tyr</td><td>235 Phe</td><td>Tyr</td><td>Val</td><td>T.rp</td><td>Leu</td><td>240 Asp</td>

<td>Ala</td><td>Pro</td><td>Ile</td><td>Gly</td><td>245 Tyr</td><td>Met</td><td>Gly</td><td>Ser</td><td>Phe</td><td>250 Lys</td><td>Asn</td><td>Leu</td><td>Cys</td><td>Asp</td><td>255 Lys</td><td>Arg</td>

<td>Gly</td><td>Asp</td><td>Ser</td><td>260 Val</td><td>Ser</td><td>Phe</td><td>Asp</td><td>Glu</td><td>265 Tyr</td><td>Trp</td><td>Lys</td><td>Lys</td><td>Asp</td><td>270 Ser</td><td>Thr</td><td>Ala</td>

<td>Glu</td><td>Leu</td><td>275 Tyr</td><td>His</td><td>Phe</td><td>Ile</td><td>Gly</td><td>280 Lys</td><td>Asp</td><td>Ile</td><td>Val</td><td>Tyr</td><td>285 Phe</td><td>His</td><td>Ser</td><td>Leu</td>

<td>Phe</td><td>290 Trp</td><td>Pro</td><td>Ala</td><td>Met</td><td>Leu</td><td>295 Glu</td><td>Gly</td><td>Ser</td><td>Asn</td><td>Phe</td><td>300 Arg</td><td>Lys</td><td>Pro</td><td>ser</td><td>A3n</td>

<td>305 Leu</td><td>Phe</td><td>Val</td><td>His</td><td>Gly</td><td>310 Tyr</td><td>Val</td><td>Thr</td><td>Val</td><td>Asn</td><td>315 Gly</td><td>Ala</td><td>Lys</td><td>Met</td><td>Ser</td><td>320 Lys</td>

<td>Ser</td><td>Arg</td><td>Gly</td><td>Thr</td><td>325 Phe</td><td>Ile</td><td>Lys</td><td>Ala</td><td>Ser</td><td>330 Thr</td><td>Trp</td><td>Leu</td><td>Asn</td><td>His</td><td>335 Phe</td><td>Asp</td>

<td>Ala</td><td>Asp</td><td>Ser</td><td>340 Leu</td><td>Arg</td><td>Tyr</td><td>Tyr</td><td>Tyr</td><td>345 Thr</td><td>Ala</td><td>Lys</td><td>Leu</td><td>Ser</td><td>350 Ser</td><td>Arg</td><td>Ile</td>

<td>Asp</td><td>Asp</td><td>355 Ile</td><td>ASp</td><td>Leu</td><td>Asn</td><td>Leu</td><td>360 Glu</td><td>Asp</td><td>Phe</td><td>Val</td><td>Gin</td><td>365 Arg</td><td>Val</td><td>Asn</td><td>Ala</td>

<td>Asp</td><td>370 Ile</td><td>Val</td><td>Asn</td><td>Lys</td><td>Val</td><td>375 Val</td><td>Asn</td><td>Leu</td><td>Ala</td><td>Ser</td><td>380 Arg</td><td>Asn</td><td>Ala</td><td>Gly</td><td>Phe</td>

<td>385 Ile</td><td>Asn</td><td>Lys</td><td>Arg</td><td>Phe</td><td>390 Asp</td><td>Gly</td><td>Val</td><td>Leu</td><td>Ala</td><td>395 Ser</td><td>Glu</td><td>Leu</td><td>Ala</td><td>Asp</td><td>400 Pro</td>

<td>Gin</td><td>Leu</td><td>Tyr</td><td>Lys</td><td>405 Thr</td><td>Phe</td><td>Thr</td><td>Asp</td><td>Ala</td><td>410 Ala</td><td>Glu</td><td>Val</td><td>ile</td><td>Gly</td><td>415 Glu</td><td>Ala</td>

<td>Trp</td><td>Glu</td><td>Ser</td><td>420 Arg</td><td>Glu</td><td>Phe</td><td>Gly</td><td>Lys</td><td>425 Ala</td><td>Val</td><td>Arg</td><td>Glu</td><td>Ile</td><td>430 Met</td><td>Ala</td><td>Leu</td>

<td>Ala</td><td>Asp</td><td>43S Leu</td><td>Ala</td><td>Asn</td><td>Arg</td><td>Tyr</td><td>440 Val</td><td>Asp</td><td>Glu</td><td>Gin</td><td>Ala</td><td>445 Pro</td><td>Trp</td><td>vai</td><td>Val</td>

<td>Ala</td><td>450 Lys</td><td>Gin</td><td>Glu</td><td>Gly</td><td>Arg</td><td>455 Asp</td><td>Ala</td><td>Asp</td><td>Leu</td><td>Giń</td><td>460 Ala</td><td>ile</td><td>Cys</td><td>Ser</td><td>Met</td>

<td>465 Gly</td><td>ILe</td><td>Asn</td><td>Leu</td><td>Phe</td><td>470 Arg</td><td>Val</td><td>Leu</td><td>Met</td><td>Thr</td><td>475 Tyr</td><td>Leu</td><td>Lys</td><td>Pro</td><td>Val</td><td>480 Leu</td>

<td>Pro</td><td>Lys</td><td>Leu</td><td>Thr</td><td>485 Glu</td><td>Arg</td><td>Ala</td><td>Glu</td><td>Ala</td><td>490 Phe</td><td>Leu</td><td>Asn</td><td>Thr</td><td>Glu</td><td>495 Leu</td><td>Thr</td>

<td>Trp</td><td>Asp</td><td>Gly</td><td>500 Ile</td><td>Gin</td><td>Gin</td><td>Pro</td><td>Leu</td><td>505 Leu</td><td>Gly</td><td>His</td><td>Lys</td><td>Val</td><td>510 Asn</td><td>Pro</td><td>Phe</td>

<td>Lys</td><td>Ala</td><td>515 Leu</td><td>Tyr</td><td>Asn</td><td>Arg</td><td>Ile</td><td>520 Asp</td><td>Met</td><td>Arg</td><td>Gin</td><td>val</td><td>525 Glu</td><td>Ala</td><td>Leu</td><td>Val</td>

<td>Glu 545</td><td>530 Ala</td><td>Ser</td><td>Lys</td><td>Glu</td><td>Glu 550</td><td>535 val</td><td></td><td></td><td></td><td></td><td> 540</td><td></td><td></td><td></td><td></td>

&lt;210&gt; 93 &lt;211&gt; 594 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 93

<td>Thr</td><td>Met</td><td>Leu</td><td>Arg</td><td>Thr</td><td>Ser</td><td>Val</td><td>Leu</td><td>Arg</td><td>Leu</td><td>Leu</td><td>Gly</td><td>Arg</td><td>Thr</td><td>Gly</td><td>Ala</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Ser</td><td>Arg</td><td>Leu</td><td>Ser</td><td>Leu</td><td>Leu</td><td>Glu</td><td>Asp</td><td>phe</td><td>Gly</td><td>Pro</td><td>Arg</td><td>Tyr</td><td>Tyr</td><td>Ser</td><td>Ser</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Gly</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Ala</td><td>Gly</td><td>Asp</td><td>Asp</td><td>Ala</td><td>Cys</td><td>Asp</td><td>Val</td><td>Arg</td><td>Ala</td><td>Tyr</td><td>Phe</td>

-170-

<td>Thr</td><td>Thr</td><td>35 Pro</td><td>Ile</td><td>Phe</td><td>Tyr</td><td>Val</td><td>40 Asn</td>

<td>Tyr</td><td>50 Ser</td><td>Ala</td><td>Leu</td><td>Leu</td><td>Ala</td><td>55 Asp</td><td>Ala</td>

<td>65 Gly</td><td>Pro</td><td>Ser</td><td>Thr</td><td>Ala</td><td>70 Ala</td><td>Thr</td><td>Arg</td>

<td>Gly</td><td>Leu</td><td>Lys</td><td>Ile</td><td>85 Gin</td><td>Gin</td><td>Ala</td><td>Ala</td>

<td>Glu</td><td>Leu</td><td>Cys</td><td>100 Asp</td><td>Arg</td><td>Val</td><td>Ser</td><td>Glu</td>

<td>Ala</td><td>Gly</td><td>115 Ile</td><td>Ser</td><td>cys</td><td>Thr</td><td>Asp</td><td>120 Phe</td>

<td>Arg</td><td>130 val</td><td>Ala</td><td>Val</td><td>Gin</td><td>His</td><td>135 phe</td><td>Trp</td>

<td>145 Leu</td><td>Tyr</td><td>Lys</td><td>Gly</td><td>Val</td><td>150 Tyr</td><td>Glu</td><td>Gly</td>

<td>Phe</td><td>Leu</td><td>Pro</td><td>Glu</td><td>165 Ala</td><td>Lys</td><td>Val</td><td>Thr</td>

<td>Ser</td><td>Phe</td><td>Pro</td><td>180 Val</td><td>Ser</td><td>Leu</td><td>Glu</td><td>Ser</td>

<td>Glu</td><td>Glu</td><td>195 Asn</td><td>Tyr</td><td>Ile</td><td>Phe</td><td>Arg</td><td>200 Leu</td>

<td>Arg</td><td>210 Trp</td><td>Leu</td><td>Arg</td><td>Gly</td><td>Asn</td><td>215 Pro</td><td>Gin</td>

<td>225 His</td><td>Val</td><td>Val</td><td>Leu</td><td>Gin</td><td>230 Trp</td><td>Leu</td><td>Asp</td>

<td>Ser</td><td>Arg</td><td>Arg</td><td>Ser</td><td>245 Ser</td><td>His</td><td>Leu</td><td>His</td>

<td>Asp</td><td>Ser</td><td>Gin</td><td>260 Thr</td><td>ile</td><td>Tyr</td><td>Val</td><td>Trp</td>

<td>Thr</td><td>Val</td><td>275 Ile</td><td>Gly</td><td>Tyr</td><td>Pro</td><td>Asn</td><td>280 Ala</td>

<td>Thr</td><td>290 Ser</td><td>Kis</td><td>Ile</td><td>Ile</td><td>Gly</td><td>295 Lys</td><td>Asp</td>

<td>305 Trp</td><td>Pro</td><td>Ala</td><td>Phe</td><td>Leu</td><td>310 Leu</td><td>Gly</td><td>Ala</td>

<td>Cys</td><td>vai</td><td>His</td><td>Ser</td><td>325 His</td><td>Trp</td><td>Thr</td><td>Val</td>

<td>Leu</td><td>Gly</td><td>Asn</td><td>340 Val</td><td>val</td><td>Asp</td><td>pro</td><td>Arg</td>

<td>Asp</td><td>Gly</td><td>355 Phe</td><td>Arg</td><td>Tyr</td><td>Phe</td><td>Leu</td><td>360 Leu</td>

<td>Cys</td><td>370 Asp</td><td>Tyr</td><td>Tyr</td><td>Asp</td><td>Glu</td><td>375 Lys</td><td>Val</td>

<td>385 Ala</td><td>Asp</td><td>Ala</td><td>Leu</td><td>Gly</td><td>3 90 Gly</td><td>Leu</td><td>Leu</td>

<td>Asn</td><td>Pro</td><td>Ser</td><td>Glu</td><td>405 Thr</td><td>Tyr</td><td>Pro</td><td>Ala</td>

<td>Glu</td><td>Pro</td><td>Gly</td><td>420 Leu</td><td>Val</td><td>Gly</td><td>Pro</td><td>Ser</td>

<td>Ala</td><td>Leu</td><td>435 Val</td><td>Ser</td><td>Ala</td><td>Val</td><td>Ala</td><td>440 Thr</td>

<td>Tyr</td><td>450 Asp</td><td>Asn</td><td>Phe</td><td>Arg</td><td>Ile</td><td>455 Tyr</td><td>Lys</td>

<td>455 Val</td><td>Arg</td><td>Gin</td><td>Thr</td><td>Asn</td><td>470 Gly</td><td>Phe</td><td>Val</td>

<td>Asn</td><td>Trp</td><td>Glu</td><td>Ser</td><td>485 Pro</td><td>Val</td><td>Asp</td><td>Ala</td>

Ala Ala Pro His Ile Gly Hi3 Leu 60

Leu Cys Arg His Arg Arg Leu Arg 75 80

Phe Ser Thr Gly Thr Asp Glu His 90 95

Ala Thr Ala Gly Leu Ala Pro Thr

105 110

Gin Phe Gin Gin Leu Phe Gin Glu ¹²⁵

Ile Arg Thr Thr Glu Ala Arg His 140

Gly val Leu Lys ser Arg Gly Leu 155 ISO

Trp Tyr Cys Ala Ser Asp Glu Cys 170 175

Gin Gin Pro Gly Pro Ser Gly Asp

185 190

Gly His Pro Val Ser Trp Thr Lys 205

Ser Gin Phe Arg Lys Pro Leu Gin 220

Ala ile Thr Pro Glu Pro Phe His 235 240

Glu Glu Leu Pro Asp Leu Ser Val 250 255

Trp Gly Ile Pro Val Pro Gly Asp

265 270

Leu Asp Ala Leu Val Asn Tyr Leu 285

Glu Phe Lys Ser Trp Trp Pro Ala 300

Ile Leu Lys Phe His Ala Ile Tyr 315 320

Gly Met Ser Pro Pro Gin Arg ile 330 335

Cys Gly Gin Lys Met Ser Lys Ser

345 350

Thr Cys Leu Asn Arg Tyr Thr Val 365

Arg Gin Gly Val Pro Asn Trp Asp 380

Val Lys Leu Leu Asn Ser Glu Leu 395 400

Asn Arg Cys Thr Ala Lys Arg Ile 410 415

Phe Cys Thr Thr Cys Phe Pro Ser

425 430

Val Arg Ala Gin Ala Glu Asp Tyr 445

Leu Pro Lys Gin Val Ala Asp His 460

Ala Leu Glu Ala Val Ser Ser cys 475 480

Gin Arg His Ala Pro Trp Lys Leu 490 495

Pro Trp Leu Gly Thr Val Leu His

<td></td><td></td><td></td><td>SOO</td><td></td><td></td><td></td><td></td>

<td>val</td><td>Ala</td><td>Leu 515</td><td>Glu</td><td>cya</td><td>Leu</td><td>Arg</td><td>val 520</td>

<td>Thr</td><td>Pro 530</td><td>Ser</td><td>Leu</td><td>Ala</td><td>Asp</td><td>Lys 535</td><td>Leu</td>

<td>Ser 54 5</td><td>Glu</td><td>Arg</td><td>Ser</td><td>Leu</td><td>Gly 550</td><td>Glu</td><td>Leu</td>

<td>His</td><td>pro</td><td>Cys</td><td>pro</td><td>Phe 565</td><td>Glu</td><td>Gly</td><td>Arg</td>

<td>Leu</td><td>Phe</td><td>Pro</td><td>Arg 580</td><td>Leu</td><td>Asp</td><td>Gin</td><td>Ser</td>

Arg Thr

<td> 505</td><td></td><td></td><td></td><td></td><td> 510</td><td></td><td></td>

<td>Phe</td><td>Gly</td><td>Thr</td><td>Leu</td><td>Leu 525</td><td>Gin</td><td>Pro</td><td>Val</td>

<td>Leu</td><td>Ser</td><td>Arg</td><td>Leu 540</td><td>Gly</td><td>Val</td><td>Ser</td><td>Ala</td>

<td>Tyr</td><td>Phe</td><td>Leu 555</td><td>Pro</td><td>Arg</td><td>Phe</td><td>Tyr</td><td>Gly 560</td>

<td>Arg</td><td>Leu 570</td><td>Gly</td><td>Pro</td><td>Glu</td><td>Thr</td><td>Gly 575</td><td>Leu</td>

<td>Arg 585</td><td>Thr</td><td>Trp</td><td>Leu</td><td>Val</td><td>Lys 590</td><td>Ala</td><td>His</td>

-171&lt;210&gt; 94 &lt;211&gt;562 &lt;212&gt; PRT &lt;213&gt; Xenopus laevis &lt;400&gt; 94

<td>Met 1</td><td>Leu</td><td>Arg</td><td>Ser</td><td>Leu 5</td><td>Ala</td><td>Leu</td><td>Arg</td><td>Thr</td><td>Phe 10</td><td>Ala</td><td>Asn</td><td>Ile</td><td>Leu</td><td>Gly 15</td><td>Leu</td>

<td>Ser</td><td>Ser</td><td>Arg</td><td>Ser 20</td><td>Gly</td><td>Ser</td><td>Thr</td><td>Ala</td><td>Ala 25</td><td>Met</td><td>Ser</td><td>Arg</td><td>Asn</td><td>Ala 30</td><td>Val</td><td>Ala</td>

<td>Ser</td><td>Arg</td><td>Pro 35</td><td>His</td><td>Leu</td><td>Tyr</td><td>Thr</td><td>Thr 40</td><td>Pro</td><td>Ile</td><td>Phe</td><td>Tyr</td><td>Val 45</td><td>Asn</td><td>Ala</td><td>Ala</td>

<td>Pro</td><td>His 50</td><td>Leu</td><td>Gly</td><td>His</td><td>Val</td><td>Tyr 55</td><td>Ser</td><td>Ala</td><td>Leu</td><td>Leu</td><td>Ala 60</td><td>Asp</td><td>val</td><td>Gln</td><td>His</td>

<td>Arg 65</td><td>Tyr</td><td>Ser</td><td>Ala</td><td>Met</td><td>Cys 70</td><td>Gly</td><td>Ile</td><td>Glu</td><td>Ser</td><td>Lye 75</td><td>Leu</td><td>Ser</td><td>Thr</td><td>Gly</td><td>Thr 80</td>

<td>Asp</td><td>Glu</td><td>His</td><td>Gly</td><td>Met 85</td><td>Lys</td><td>Val</td><td>Gln</td><td>Gln</td><td>Ala 90</td><td>Ala</td><td>Ser</td><td>Ala</td><td>Leu</td><td>Gly 95</td><td>Leu</td>

<td>Asp</td><td>pro</td><td>Gln</td><td>Thr 100</td><td>Phe</td><td>Cys</td><td>Ser</td><td>Thr</td><td>Val 105</td><td>Ser</td><td>Leu</td><td>Gln</td><td>Phe</td><td>Arg 110</td><td>Thr</td><td>Ile</td>

<td>Phe</td><td>Asp</td><td>Ala 115</td><td>Leu</td><td>Asp</td><td>Ile</td><td>Ser</td><td>Tyr 120</td><td>Thr</td><td>Asp</td><td>Phe</td><td>val</td><td>Arg 125</td><td>Thr</td><td>Thr</td><td>Glu</td>

<td>Pro</td><td>Arg 130</td><td>His</td><td>Ile</td><td>Glu</td><td>Ala</td><td>Val 135</td><td>Ser</td><td>Arg</td><td>Phe</td><td>Trp</td><td>Met 140</td><td>Thr</td><td>Leu</td><td>Glu</td><td>Glu</td>

<td>Gln 145</td><td>Gly</td><td>Tyr</td><td>Ile</td><td>Tyr</td><td>Lys 150</td><td>Gly</td><td>Thr</td><td>Tyr</td><td>Glu</td><td>Gly 155</td><td>Trp</td><td>Tyr</td><td>Cys</td><td>Thr</td><td>Ser 160</td>