Genetically programmed expression of selectively sulfated proteins in eubacteria

Genetically programmed expression of selectively sulfated proteins in eubacteria

5 Field of the Invention

The invention belongs to the field of translation biochemistry. The invention relates to compositions and methods for manufacturing and using orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and pairs thereof, which incorporate unnatural amino acids into proteins. The invention also relates to methods for producing proteins in cells using said pairs and proteins manufactured by the methods.

Background of the invention

Sulfation of tyrosine is a common post-translational modification in segregated and protein-bound proteins.

fifteen membranes (Kehoe and Bertozzi, "Tyrosine sulfation: a modulator of extracellular protein-protein interactions," Chem Biol 7: R57-61 (2000)). Although we are only beginning to understand the degree of its biological function, sulfotyrosine has already been identified in various protein-protein interaction paradigms. For example, tyrosine sulfation plays a determining role in chemokine binding to CCR2 chemokine receptors (Preobrazhensky et al., “Monocyte chemotactic protein-1 CCR2B receptor is a glycoprotein that has tyrosine sulfation in a conserved extracellular N- terminal region ”J Immunol 165: 5295-5303 (2000)), CCR5 (Farzan et al.,“ Tyrosine sulfation of the amino terminus of CCR5 facilitates HIV-1 entry ”Cell 96: 667-676 (1999)), CXCR4 (Farzan et al., “The role of post-translational modifications of the CXCR4 amino terminus in stromal-derived factor 1 alpha association and HIV-1 entry,” J Biol Chem 277: 29484- 29489 (2002); Veldkamp et al., "Recognition of a CXCR4 sulfotyrosine by the chemokine stromal cell-derived factorlalpha (SDF-1alpha / CXCL12)," J Mol Biol 359:

25 1400-1409 (2006)) and CX3CR1 (Fong et al., "CX3CR1 tyrosine sulfation enhances fractalkine-induced cell adhesion," J Biol Chem 277: 19418-19423 (2002)). Similarly, rolling leukocytes under hydrodynamic shear stress require sulfation of PSGL-1 for proper binding and adhesion (Somers et al., “Insights into the molecular basis of leukocyte tethering and rolling revealed by structures of P- and E-selectin bound to SLe (X) and PSGL-1, ”Cell 103: 467-479 (2000)). Sulfation of tyrosine is also involved in the coagulation cascade, having been identified in various coagulation factors as well as in natural thrombin inhibitors such as hirudin, a blood sugar-secreted anticoagulant (Dong et al., “Tyrosine sulfation of the glycoprotein Ib-IX complex: identification of sulfated residues and effect on ligand binding, ”Biochemistry 33: 13946-13953 (1994); Bagdy et al.,“ Hirudin, ”Methods Enzymol 45: 669-678 (1976)). In addition, it was recently discovered that sulfation of tyrosine in a variable loop region of antibody was responsible for the neutralizing activity of

35 a subset of VHI-1 antibodies induced by CD4, thereby demonstrating the ability of sulfotyrosine to increase antibody antigen affinity (Choe et al., “Tyrosine sulfation of human antibodies contributes to recognition of the CCR5 binding region of HIV- 1 gp120, "Cell 114: 161-170 (2003); Xiang et al.," Functional mimicry of a human immunodeficiency virus type 1 coreceptor by a neutralizing monoclonal antibody, "J Virol 79: 6068-6077 (2005)).

A major obstacle to the determination of sulfation functions in more than 60 known proteins and more than 2100 predicted sulfotyrosine-containing proteins (according to a study of mouse protein sequences) is the ability to selectively synthesize sulfated proteins (Moore , "The biology and enzymology of protein tyrosine O-sulfation," J Biol Chem 278: 24243-24246 (2003)). Current methods are based on synthesis

Four. Five of conventional or in vitro enzymatic sulfation peptides (Veldkamp et al., "Recognition of a CXCR4 sulfotyrosine by the chemokine stromal cell-derived factor-lalpha (SDF-1alpha / CXCL12)," J Mol Biol 359: 1400-1409 (2006 ); Kirano et al., "Total synthesis of porcine cholecystokinin-33 (CCK-33)," J. Chem. Soc., Chem. Com-mun., 323-325 (1987); Muramatsu et al., "Enzymic O-sulfation of tyrosine residues in hirudins by sulfotransferase from Eubacterium A-44," Eur J Biochem 223: 243-248 (1994); Young and Kiessling, "A strategy for the synthesis of sulfated peptides," Angew Chem Int Ed Engl 41: 3449-3451 (2002)); however, both lack generalities: the first is limited by restrictions of length and the tendency towards desulphation of sulfothyrosine under acidic conditions; the latter is limited by the availability of accessory sulfotransferases and their associated recognition sequence restrictions.

55 The direct incorporation of an unnatural amino acid of genetically encoded sulfothyrosine into protein-defined sites directly in living organisms would overcome the limitations described above. The direct incorporation of sulfothyrosine would greatly facilitate the study of sulfation events in the regulation of biological processes and also allow the creation of libraries of sulfated antibodies and peptides of significant diversity. Additionally, the ability to produce a sulphated form in the hirudin protein has immediate clinical application for use as an improved anticoagulant (improved with respect to the non-sulphated form). What is needed in the art are new strategies to incorporate the unnatural amino acid sulfotyrosine into proteins.

A general methodology has been developed for site specific in vivo incorporation of various

65 unnatural amino acids in proteins, in both eukaryotic and prokaryotic organisms. These methods are based on orthogonal protein translation components that recognize a selector codon suitable for inserting a desired unnatural amino acid at a defined position during polypeptide translation in vivo. These methods use an orthogonal tRNA (O-tRNA) that recognizes a selector codon, and in which a corresponding orthogonal aminoacyl-tRNA synthetase (an O-RS) loads the O-tRNA with the unnatural amino acid. These components do not cross-react with any of the tRNAs, RSs, amino acids or endogenous codons in the

5 host organism (that is, it must be orthogonal). The use of such orthogonal tRNA pairs has made it possible to genetically encode a large number of structurally diverse non-natural amino acids.

The practice of using orthogonal translation systems that are suitable for the manufacture of proteins comprising one or more unnatural amino acids is generally known in the art, as are the general methods for producing orthogonal translation systems. For example, see International Publication Numbers WO 2002/086075, entitled "METHODS AND COMPOSITION FOR THE PRODUCTION OF ORTHOGONAL tRNA-AMINOACYL-tRNA SYNTHETASE PAIRS"; WO 2002/085923, entitled "IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS"; WO 2004/094593, entitled "EXPANDING THE EUKARYOTIC GENETIC CODE"; WO 2005/019415, filed July 7, 2004; WO 2005/007870, filed July 7, 2004; WO 2005/007624, filed on July 7, 2004 and WO 2006/110182, filed on October 27, 2005, entitled “ORTHOGONAL TRANSLATION COMPONENTS FOR THE VIVO INCORPORATION OF UNNATURAL AMINO ACIDS”. For further analysis of orthogonal translation systems that incorporate unnatural amino acids, and methods for their production and use, see also, Wang and Schultz, "Expanding the Genetic Code," Chem. Commun. (Camb.) 1: 1-11 (2002); Wang and Schultz “Expanding the Genetic Code,” 20 Angewandte Chemie Int. Ed., 44 (1): 34-66 (2005); Xie and Schultz, "An Expanding Genetic Code," Methods 36 (3): 227238 (2005); Xie and Schultz, "Adding Amino Acids to the Genetic Repertoire," Curr. Opinion in Chemical Biology 9 (6): 548-554 (2005); Wang et al., "Expanding the Genetic Code," Annu. Rev. Biophys. Biomol Struct., 35: 225-249 (2006; epub June 13, 2006); and Xie and Schultz, "A chemical toolkit for proteins -an expanded genetic code," Nat. Rev. Mol. Cell Biol., 7 (10): 775-782 (2006; electronic publication August 23, 2006). WO 2004/035743

25 describes orthogonal tRNA / aminoacyl tRNA synthetase pairs that incorporate keto amino acids into proteins.

There is a need in the art to develop orthogonal translation components that incorporate the unnatural amino acid sulfothyrosine into proteins, in which the unnatural amino acid can be incorporated in any defined position. The invention described herein meets these and other needs, as will be appreciated.

30 after review of the following disclosure.

Summary of the invention

Although tyrosine sulphation is a generalized post-translational modification in multicellular eukaryotes.

35 (Moore, "The biology and enzymology of protein tyrosine O-sulfation," J Biol Chem 278: 24243-24246 (2003)), its biological functions remain largely unknown. This is due in part to the difficulties associated with the synthesis of selectively sulfated proteins. The invention provides the selective incorporation of sulfothyrosine into proteins in bacteria by genetic coding of the modified amino acid in response to the amber nonsense codon, TAG. In addition, it is shown that sulfo-hirudin, previously inaccessible through methods

40 recombinants, can be expressed directly in E. coli using this strategy. As described herein, kinetic analyzes show an improvement in affinity greater than 10 times against human thrombin by sulfohirudin over desulfohirudin, an observation that offers clinical advantages for sulfohirudin in its use as an anticoagulant (Di Nisio et al ., "Direct thrombin inhibitors," N Engl J Med 353: 10281040 (2005)). This general strategy for the biosynthesis of sulfated proteins facilitates the study and application

Four. Five Additional post-translational modification emergent, sulfation of tyrosine.

The invention provides compositions and methods, as defined in the claims, for the in vivo incorporation (eg, into a host cell) of the unnatural amino acid sulfothyrosine into a growing polypeptide chain in response to a selector codon, for example, a amber termination codon. These compositions include orthogonal tRNA pairs (O-tRNA) and orthogonal aminoacyl-tRNA synthetases (O-RSs) that do not interact with the host cell translation machinery. That is, the O-tRNA is not charged (or not charged at a significant level) with an amino acid (natural or unnatural) by an aminoacyl-tRNA synthetase of the endogenous host cell. Similarly, the O-RSs provided by the invention do not load any endogenous tRNA with an amino acid (natural or unnatural) at a significant or detectable level. These new compositions

55 they allow the production of large amounts of proteins that have translationally incorporated sulfothyrosine.

In a first aspect, the invention provides translation systems as defined in the claims. Translation systems comprise: (a) a first unnatural amino acid that is sulfothyrosone as depicted in Figure 1; (b) a first orthogonal aminoacyl tRNA aminoacyl (O-RS) comprising a 60 amino acid sequence that is a conservative variant of SEQ ID NO: 2, whose conservative variant is at least 90% identical to SEQ ID NO: 2 and comprises: a leucine in a position corresponding to Tyr32; a proline in a position corresponding to Leu65; a glycine in a position corresponding to Asp158, and a lysine in a position corresponding to Leu162, and; optionally it comprises a glutamic acid in a position corresponding to Gln155, and / or a threonine or a cysteine ​​in a position corresponding to Ile159, and; (c) a first orthogonal tRNA

65 (O-tRNA); wherein the first O-RS aminoacylates the first O-tRNA with sulfothyrosine.

In certain embodiments, the first O-RS is derived from a methanococcus jannaschii aminoacyl-tRNA synthetase, and / or derived from a wild-type Methanococcus jannaschii tyrosyl tRNA synthetase and / or comprises an amino acid sequence set forth in SEQ ID Nos: 4, 6, 8 or 10. The first O-tRNA may be an amber suppressor tRNA and / or may comprise or be encoded by a polynucleotide sequence set forth in SEQ ID NO:

5 1.

In certain embodiments, the translation system further comprises a nucleic acid encoding a protein of interest. The nucleic acid comprises at least one selector codon that recognizes the first O-tRNA. The translation system may additionally comprise a second O-RS and a second O-tRNA, wherein the second O-RS aminoacylates the second O-tRNA with a second unnatural amino acid that is different from the first unnatural amino acid, and in that the second O-tRNA recognizes a selector codon that is different from the selector codon recognized by the first O-tRNA.

In certain embodiments, the translation system comprises a host cell comprising the

fifteen first unnatural amino acid, the first O-RS and the first O-tRNA. The host cell may be a eubacterial cell, such as an E. coli cell. The host cell may comprise a polynucleotide encoding the first O-RS and / or a polynucleotide encoding the first O-tRNA. The polynucleotide encoding the first O-RS may comprise a nucleotide sequence set forth in SEQ ID Nos: 5, 7, 9 or 11.

In a second aspect, the invention provides methods for producing, in a translation system, a protein comprising an unnatural amino acid in a selected position as defined in the claims. The method comprises: (a) providing a translation system comprising: (i) a first unnatural amino acid that is sulfothyrosine as depicted in Fig. 1; (ii) a first O-RS comprising the amino acid sequence that is a conservative variant of SEQ ID NO: 2, whose conservative variant is at least 90%

25 identical to SEQ ID NO: 2 and comprises: a leucine in a position corresponding to Tyr32; a proline in a position corresponding to Leu65; a glycine in a position corresponding to Asp158, and a lysine in a position corresponding to Leu162, and; optionally it comprises a glutamic acid in a position corresponding to Gln155, and / or a threonine or a cysteine ​​in a position corresponding to Ile159; (iii) a first O-tRNA, in which the first O-RS aminoacylates the first O-tRNA with sulfotyrosine; and, (iv) a nucleic acid encoding the protein, wherein the nucleic acid comprises at least one selector codon recognized by the first O-tRNA, and; (b) incorporating the unnatural amino acid at the selected position in the protein during translation of the protein in response to the selector codon, thereby producing the protein comprising the unnatural amino acid at the selected position.

35 In certain embodiments of the methods, the protein comprising an unnatural amino acid is sulfohirudin.

In certain embodiments of the methods, providing a translation system may comprise: (A) providing a polynucleotide encoding the O-RS or (B) providing an O-RS derived from a methanococcus jannaschii aminoacyl-tRNA synthetase, or; (C) provide an O-RS derived from a wild-type Methanococcus jannaschii tyrosyl-tRNA synthetase, or; (D) provide an O-RS comprising an amino acid sequence set forth in SEQ ID Nos: 4, 6, 8 or 10, or; (E) mutating an amino acid binding pocket of a wild-type aminoacyl-tRNA synthetase by site-directed mutagenesis and selecting a resulting O-RS that aminoacile said O-tRNA with said unnatural amino acid, in which the selection step comprises positively selecting and negatively selecting the O-RS from a set comprising a plurality of aminoacyl-tRNA synthetase molecules resulting after site directed mutagenesis, or; (F) providing a polynucleotide encoding said O-tRNA, or; (G) provide an O-tRNA that is an amber suppressor tRNA, or,

(H) provide an O-tRNA that comprises or is encoded by a polynucleotide sequence set forth in SEQ ID NO: 1, or; (I) provide a nucleic acid comprising an amber selector codon, or; (J) providing a host cell comprising the first non-natural amino acid, the first O-RS, the first O-tRNA and the nucleic acid, and the incorporation step comprises culturing the host cell, or; (K) provide a cell extract.

In certain embodiments of (J), providing a host cell comprises providing a cell.

55 Eubacteria host, which may be an E. coli host cell, or provide a host cell comprising a polynucleotide encoding the O-RS, which may comprise a nucleotide sequence set forth in SEQ ID Nos: 5, 7, 9 or 11.

In certain embodiments of the methods, the protein comprises a second non-natural amino acid that is different from the first non-natural amino acid, and the translation system further comprises a second O-RS and a second O-tRNA. The second O-RS aminoacylates the second O-tRNA with a second unnatural amino acid that is different from the first unnatural amino acid, and the second O-tRNA recognizes a selector codon in the nucleic acid that is different from the selector codon recognized by the first O-tRNA

65 In a third aspect, the invention provides compositions comprising a polypeptide, which comprises an amino acid sequence that is a conservative variant of SEQ ID NO: 2, whose conservative variant is at least 90% identical to SEQ ID NO: 2 , and comprises: a leucine in a position corresponding to Tyr32; a proline in a position corresponding to Leu65; a glycine in a position corresponding to Asp158 and a lysine in a position corresponding to Leu162, and; optionally it comprises a glutamic acid in a position corresponding to Gln155, and / or a threonine or a cysteine ​​in a position corresponding to Ile159; whose

5 aminoacyla polypeptide an O-tRNA with sulfothyrosine as depicted in Fig. 1.

In certain embodiments of the compositions, the polypeptide comprises the amino acid sequence of SEQ ID Nos: 4, 6, 8 or 10. In certain embodiments, the compositions comprise a cell comprising the polypeptide.

10 In a related aspect, the invention provides polynucleotides encoding the polypeptides of the compositions. In certain embodiments, the polynucleotides comprise the nucleotide sequence SEQ ID Nos: 5, 7, 9 or 11.

fifteen In a further related aspect, the invention provides vectors comprising the polynucleotide encoding the polypeptide of the compositions.

In a further related aspect, the invention provides cells comprising the vectors.

twenty In some aspects, the translation system incorporates a second orthogonal pair (i.e., a second O-RS and a second O-tRNA) that uses a second unnatural amino acid, so that the system can now incorporate at least two amino acids unnatural at different sites selected in a polypeptide. In this double system, the second O-RS preferably aminoacylates the second O-tRNA with a second unnatural amino acid that is different from the first unnatural amino acid, and the second O-tRNA recognizes a selector codon that is

25 different from the selector codon recognized by the first O-tRNA.

In some embodiments, the translation system resides in a host cell (and includes the host cell). The host cell used is not particularly limited, provided that the O-RS and the O-tRNA retain their orthogonality in their host cell medium. The host cell can be a cell of

30 eubacteria, such as E. coli. The host cell may comprise one or more polynucleotides that encode components of the translation system, including the O-RS or the O-tRNA. In some embodiments, the polynucleotide encoding the O-RS comprises a nucleotide sequence of SEQ ID Nos: 5, 7, 9 or 11.

The invention also provides methods for producing proteins that have one or more unnatural amino acids.

35 in selected positions. These methods use the translation systems described above. Generally, these methods begin with the step of providing a translation system comprising: (i) a first unnatural amino acid that is the unnatural amino acid sulfothyrosine; (ii) a first orthogonal aminoacyl-tRNA synthetase (O-RS); (iii) a first orthogonal tRNA (O-tRNA), in which the O-RS preferably aminoaclates the O-tRNA with the unnatural amino acid; and, (iv) a nucleic acid encoding the protein, in which the nucleic acid

40 It comprises at least one selector codon recognized by the first O-tRNA. Thus the method incorporates the unnatural amino acid at the selected position in the protein during translation of the protein in response to the selector codon, thereby producing the protein comprising the unnatural amino acid at the selected position. In some aspects of these methods, the O-RS preferably aminoacylates the O-tRNA with the sulfothyrosine with an efficiency that is at least 50% of the observed efficiency for a translation system that

Four. Five it comprises the same O-tRNA, sulfotyrosine, and an aminoacyl-tRNA synthetase comprising the amino acid sequence of SEQ ID Nos: 4, 6, 8 or 10. In some aspects, the methods are used to produce the sulfated form of the hirudin

These methods can be widely applied using various reagents and steps. In some embodiments, it

fifty provides a polynucleotide that encodes the O-RS. In some embodiments, an O-RS derived from a methanococcus jannaschii aminoacyl-tRNA synthetase is provided, for example, a wild-type Methanococcus jannaschii tyrosyl tRNA synthetase can be provided. In some embodiments, the step of providing includes providing an O-RS comprising an amino acid sequence of SEQ ID Nos: 4, 6, 8 or 10, and conservative variants thereof.

55 In some embodiments of these methods, providing a stage of the translation system comprises mutating an amino acid binding pocket of a wild-type aminoacyl-tRNA synthetase by site directed mutagenesis, and selecting a resulting O-RS that preferentially aminoacils the O -RNA with the unnatural amino acid. The selection stage may comprise selecting positively and negatively the O-RS of a set of

60 aminoacyl-tRNA synthetase molecules resulting after site-directed mutagenesis. In some embodiments, the step of providing provides a polynucleotide encoding the O-tRNA, for example, an O-tRNA that is an amber suppressor tRNA, or an O-tRNA that comprises, or is encoded by, a SEC polynucleotide. ID Nº: 1. In these methods, the step of providing can also supply a nucleic acid comprising an amber selector codon that is used by the translation system.

65 These methods can also be modified to incorporate more than one unnatural amino acid into a protein. In these methods, together with the first translation system a second orthogonal translation system is used, in which the second system has different specificities of selector codons and amino acids. For example, the step of providing may include providing a second O-RS and a second O-tRNA, in which the second

5 O-RS preferably aminoacylates the second O-tRNA with a second unnatural amino acid that is different from the first unnatural amino acid, and in which the second O-tRNA recognizes a selector codon in the nucleic acid that is different from the selector codon recognized by the first O-tRNA.

Methods for producing a protein with an unnatural amino acid can also be performed in the context of a host cell. In these cases, a host cell is provided, in which the host cell comprises the unnatural amino acid, the O-RS, the O-tRNA and the nucleic acid with at least one selector codon encoding the protein, and in which Host cell culture results in the incorporation of the unnatural amino acid. In some embodiments, the step of providing comprises providing a eubacteria host cell (eg, E. coli). In some embodiments, the stage of providing includes

fifteen provide a host cell that contains a polynucleotide encoding the O-RS. For example, the polynucleotide encoding the O-RS may comprise a nucleotide.

Definitions

Before describing the invention in detail, it should be understood that the present invention is not limited to particular biological systems that, of course, may vary. It should also be understood that the terminology used in this document has the sole purpose of describing particular embodiments and is not intended to be limiting. As used in this specification and in the appended claims, the singular forms "a", "a", "the" and "the" include plurals, unless the context clearly indicates otherwise. Therefore, the reference, by

25 example, a "one cell" includes combinations of two or more cells; the reference to "a polynucleotide" includes, as a practical matter, many copies of that polynucleotide.

Unless defined in this document and later in the rest of the specification, all scientific and technical terms used in this document have the same meaning as normally understood by a person skilled in the art to which it belongs. the invention.

Orthogonal: As used herein, the term "orthogonal" refers to a molecule (for example, an orthogonal tRNA (O-tRNA) and / or an orthogonal aminoacyl-tRNA synthetase (O-RS)) that works with Endogenous components of a cell with less efficiency compared to a corresponding molecule that is endogenous to the cell or the translation system, or fails to function with endogenous components of the cell. In the context of tRNAs and aminoacyl-tRNA synthetases, orthogonal refers to disability or lower efficacy, for example, a reduced efficiency of 20%, less than 10%, less than 5%, or less than a 1% of an orthogonal tRNA to function with an endogenous tRNA synthetase compared to the ability of an endogenous tRNA to function with the endogenous tRNA synthetase, or of an orthogonal aminoacyl tRNA synthetase to function with an endogenous tRNA compared to the ability of an endogenous tRNA synthetase to function with the endogenous tRNA. The orthogonal molecule lacks a functionally normal endogenous complementary molecule in the cell. For example, an orthogonal tRNA in a cell is aminoacylated by any endogenous RS of the cell with reduced or even zero efficiency compared to the aminoacylation of an endogenous tRNA by the endogenous RS. In another example, an orthogonal RS aminoacylates any endogenous tRNA in a cell of interest with a

Four. Five reduced or even zero efficacy, compared to the aminoacylation of endogenous tRNA by endogenous RS. A second orthogonal molecule that works with the first orthogonal molecule can be introduced into the cell. For example, an orthogonal tRNA / RS pair includes complementary components introduced that work together in the cell with efficacy (for example, an efficiency of 45%, 50%, 60%, 70%, 75%, 80%, 90% , 95% or 99% or greater) compared to that of a control, for example, a corresponding endogenous tRNA / RS pair, or an active orthogonal pair (for example, an orthogonal tRNA / RS tyrosyl pair).

Orthogonal tyrosyl-tRNA: As used herein, an orthogonal tyrosyl-tRNA (tyrosyl-O-tRNA) is an tRNA that is orthogonal to a translation system of interest, in which the tRNA is: (1) identical or substantially similar to a tyrosyl tRNA of natural origin, (2) derived from a tyrosyl tRNA of natural origin by natural or artificial mutagenesis, 55 (3) derived by any process that takes into account a wild-type tyrosyl-tRNA sequence or mutant of

(1) or (2), (4) homologous to a wild or natural tyrosyl-tRNA; (5) Homologous to any example tRNA that is designed as a substrate for a tyrosyl tRNA synthetase in FIG. 7 or (6) a conservative variant of any example tRNA that is designed as a substrate for a tyrosyl-tRNA synthetase in FIG. 7. Tyrosyl-tRNA may exist loaded with an amino acid, or in an uncharged state. It should also be understood that a "tyrosyl-O-tRNA" is optionally charged (aminoacylated) by a related synthetase with an amino acid other than tyrosine, respectively, for example, with an unnatural amino acid. In fact, it will be appreciated that a tyrosyl-O-tRNA of the present disclosure is advantageously used to insert essentially any amino acid, whether it is natural or unnatural, into the growing polypeptide, during translation, in response to a selector codon.

65 Orthogonal Tyrosyl Amino Acid Synthetase: As used herein, an orthogonal tyrosyl amino acid synthetase (tyrosyl-O-RS) is an enzyme that preferably aminoacylates tyrosyl-O-tRNA with an amino acid in a translation system of interest. The amino acid that tyrosyl-O-RS carries on tyrosyl-O-tRNA can be any amino acid, whether it is natural or unnatural or artificial and is not limited herein. The synthetase is optionally the same as or homologous to a naturally occurring tyrosyl amino acid synthetase, or the same or homologous to a synthetase designed as O-RS in FIG. 7. For example, the O-RS can be a variant

5 conservative of a tyrosyl-O-RS of FIG. 7, and / or may have an identity of at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more with an O-RS of FIG. 7.

Affine: The term "affine" refers to components that work together, or that have some aspect of specificity to each other, that is, an orthogonal tRNA and an orthogonal aminoacyl-tRNA synthetase. The components can also be called complementary.

Aminoacyla preferably: as used herein in reference to orthogonal translation systems, an O-RS "preferably aminoacylates" a related O-tRNA when the O-RS loads an O-tRNA with an amino acid more efficiently than that loads any endogenous tRNA into an expression system. That is to say,

fifteen When the O-tRNA and any given endogenous tRNA are present in a translation system at approximately the same molar proportions, the O-RS will load the O-tRNA more frequently than the endogenous tRNA will load. Preferably, the relative proportion of the O-tRNA charged by the O-RS with respect to the endogenous tRNA loaded by the O-RS is high, preferably resulting in the O-RS loading the O-tRNA exclusively, or almost exclusively, when O-tRNA and an endogenous tRNA are present at the same molar concentrations in the translation system. The relative ratio between the O-tRNA and the endogenous tRNA that is loaded by the O-RS, when the O-tRNA and the O-RS are present at the same molar concentrations, is greater than 1: 1, preferably at least approximately 2: 1, more preferably 5: 1, even more preferably 10: 1, even more preferably 20: 1, even more preferably 50: 1, even more preferably 75: 1, even more preferably 95: 1, 98: 1, 99 : 1, 100: 1, 500: 1, 1,000: 1, 5,000: 1 or greater.

25 The O-RS "preferably aminoacylates an O-tRNA with a natural amino acid" when (a) the O-RS preferably aminoacylates the O-tRNA compared to an endogenous tRNA and (b) when the aminoacylation is specific for the unnatural amino acid , in comparison with the aminoacylation of the O-tRNA by the O-RS with a natural amino acid. That is, when unnatural and natural amino acids are present in the same molar amounts in a translation system comprising the O-RS and an O-tRNA, the O-RS will load the O-tRNA with the unnatural amino acid more frequently. than with the natural amino acid. Preferably, the relative proportion of O-tRNA loaded with the unnatural amino acid with respect to the O-tRNA loaded with the natural amino acid is high. More preferably, the O-RS loads the O-tRNA exclusively, or almost exclusively, with the unnatural amino acid. The relative ratio between the loading of the O-tRNA with the non-natural amino acid and the loading of the O-tRNA with the

35 Natural amino acid, when both natural and non-natural amino acids are present in the translation system at the same molar concentrations, it is greater than 1: 1, preferably at least about 2: 1, more preferably 5: 1, even more preferably 10 : 1, even more preferably 20: 1, even more preferably 50: 1, even more preferably 75: 1, even more preferably 95: 1, 98: 1, 99: 1, 100: 1, 500: 1, 1,000: 1 , 5,000: 1 or higher.

Selector codon: the term "selector codon" refers to codons recognized by the O-tRNA in the translation process and not recognized by an endogenous tRNA. The O-tRNA anticodon loop recognizes the selector codon in the mRNA and incorporates its amino acid, for example, an unnatural amino acid, at this site in the polypeptide. Selector codons may include, for example, nonsense codons, such as, termination codons, by

Four. Five example, amber, ocher and opal codons; codons of four or more bases, rare codons; codons derived from natural or unnatural base pairs and / or the like.

Suppressor tRNA: a suppressor tRNA is an tRNA that alters the reading of a messenger RNA (mRNA) in a particular translation system, normally allowing the incorporation of an amino acid in response to a termination codon (ie, "complete reading") during the translation of a polypeptide. In some aspects, a selector codon of the invention is a suppressor codon, for example, a termination codon (for example, an amber, ocher or opal codon), a four base codon, a rare codon, etc.

Suppression activity: as used herein, the term "suppression activity" refers, in

55 In general, the ability of an tRNA (for example, a suppressor tRNA) to allow complete translation reading of a codon (for example, a selector codon that is an amber codon or a codon of four or more bases) than another This would result in the termination of the translation or the erroneous translation (for example a reading phase shift). The suppression activity of a suppressor tRNA can be expressed as a percentage of the full translation read activity observed in comparison to a second suppressor tRNA or in comparison to a control system, for example, a control system lacking an O -RS.

The present description provides various methods by which suppression activity can be quantified. The percentage of suppression of a particular O-tRNA and an O-RS against a selector codon (for example, an amber codon) of interest refers to the percentage of activity of a given expressed test marker (e.g., LacZ), which includes a selector codon, in a nucleic acid encoding the expressed test marker, in a translation system of interest, in which the translation system of interest includes a

O-RS and an O-tRNA, compared to a positive control construct, in which the positive control lacks the O-tRNA, the O-RS and the selector codon. Therefore, for example, if an active positive control marker construct lacking a selector codon has an observed activity of X in a given translation system, in units relevant to the marker assay in question, then the suppression percentage from

5 An assay construct comprising the selector codon is the percentage of X that shows the assay marker construct essentially under the same conditions as the natural environment in which the positive control marker was expressed, except that the assay marker construct It is expressed in a translation system that also includes the O-tRNA and the O-RS. Normally, the translation system that expresses the assay marker also includes an amino acid that is recognized by the O-RS and the O-tRNA. Optionally, the suppression percentage measurement can be refined by comparing the test marker with a "background" or "negative" control marker construction, which includes the same selector codon as the test marker, but in a system that does not includes the O-tRNA, the O-RS and / or the relevant amino acid recognized by the O-tRNA and / or the O-RS. This negative control is useful for normalizing the suppression percentage measurements to take into account the background signal effects of the marker in the translation system of interest.

fifteen The suppression efficiency can be determined by any of several assays known in the art. For example, a β-galactosidase indicator assay can be used, for example, a derivatized lacZ plasmid (in which the construct has a selector codon in the lacZ nucleic acid sequence) is introduced into cells of an appropriate organism (e.g., an organism in which orthogonal components can be used) together with the plasmid comprising an O-tRNA of the invention. A related synthetase can also be introduced (either as a polypeptide or as a polynucleotide encoding the related synthetase when expressed). The cells are grown in medium to a desired density, for example, up to an OD600 of about 0.5, and β-galactosidase assays are performed, using, for example, the BetaFluor ™ β-galactosidase Assay Kit (Novagen) . The percentage of suppression can be calculated as the percentage of activity for a sample with respect to a

25 comparable control, for example, the observed value for the derivatized lacZ construction, in which the construction has a corresponding sense codon in a desired position instead of a selector codon.

Translation system: the term "translation system" refers to components that incorporate an amino acid into a growing polypeptide chain (protein). Components of a translation system may include, for example, ribosomes, tRNA, synthetases, mRNA and the like. The O-tRNA and / or the O-RS of the invention can be added or part of an in vitro or in vivo translation system, for example, in a non-eukaryotic cell, for example, a bacterium (such as E. coli) or in a eukaryotic cell, for example, a yeast cell, a mammalian cell, a plant cell, an algae cell, a fungus cell, an insect cell and / or the like.

35 Unnatural amino acid: As used herein, the term "unnatural amino acid" refers to any amino acid, modified amino acid and / or amino acid analog, which is not one of the 20 common naturally occurring amino acids or natural amino acids. rare selenocysteine ​​or pyrrolysine. For example, the unnatural amino acid sulfothyrosine; see FIG. 1) find use with the invention.

Derived from: As used herein, the term "derivative of" refers to a component that is isolated or manufactured using a specified molecule or organism, or information from the specified molecule or organism. For example, a polypeptide derived from a second polypeptide may include an amino acid sequence that is identical or substantially similar to the amino acid sequence of the second polypeptide. In the case of polypeptides, the derived species can be obtained, for example, by naturally occurring mutagenesis,

Four. Five artificial directed site mutagenesis or artificial random mutagenesis. The mutagenesis used to derive polypeptides can be intentionally directed or intentionally random, or a mixture of each. The mutagenesis of a polypeptide to create a different polypeptide derived from the first may be a random event (for example, produced by polymerase infidelity) and the identification of the derived polypeptide can be performed by appropriate scanning methods, for example, as discussed in This document. Mutagenesis of a polypeptide usually involves manipulation of the polynucleotide encoding the polypeptide.

Selection marker or positive identification: as used herein, the term "selection marker or positive identification" refers to a marker which, when present, for example, expressed, activated or similar, results in the identification of a cell comprising the feature, for example, a cell

55 with the positive selection marker, among those that do not have the trait.

Selection or negative identification marker: as used herein, the term "selection or negative identification marker" refers to a marker which, when present, for example expressed activated or similar, allows the identification of a cell that It does not comprise a selected property or feature (for example, compared to a cell that has the property or feature).

Indicator: as used herein, the term "indicator" refers to a component that can be used to identify and / or select target components of a system of interest. For example, an indicator may include a protein, for example, an enzyme, which confers resistance or sensitivity to antibiotics (for example, β-lactamase, chloramphenicol acetyltransferase (CAT) and the like), a fluorescent identification marker (e.g., green fluorescent protein (for example, (GFP), YFP, EGFP, RFP, etc.), a luminescent marker (for

example a firefly luciferase protein), an affinity-based identification marker, or positive or negative selection marker genes, such as lacZ, β-gal / lacZ β-galactosidase), ADH (alcohol dehydrogenase), his3, ura3, leu2, lys2 or the like.

5 Eukaryotic: as used herein the term "eukaryotic" refers to organisms that belong to the Eucarya Kingdom. Eukaryotes generally differ from prokaryotes by their normally multicellular organization (but not exclusively multicellular, for example, yeast), the presence of a nucleus bounded by a membrane and other organelles bounded by a membrane, linear genetic materials (for example, chromosomes linear), the absence of operons, the presence of introns, mRNA with message protection and poly-A and other biochemical characteristics, such as a differentiable ribosomal structure. Eukaryotic organisms include, for example, animals (for example, mammals, insects, reptiles, birds, etc.), ciliates, plants (for example, monocots, dicotyledons, algae, etc.), fungi, yeasts, flagellate, microsporidia, protists, etc.

Prokaryotic: as used herein, the term "prokaryotic" refers to organisms that belong

fifteen to the Monera Kingdom (also called Procarya). Prokaryotic organisms generally differ from eukaryotes by their unicellular organization, asexual reproduction by budding or fission, the absence of a nucleus bounded by a membrane or other organelles bounded by a membrane, a circular chromosome, the presence of operons, the absence of introns, message protection and poly-A mRNA and other biochemical characteristics such as a differentiable ribosomal structure. Prokaryotes include the subbalances Eubacteria and Archaea (sometimes referred to as "Archaebacteria"). Cyanobacteria (blue-green algae) and mycoplasmas sometimes occur in different classifications in the Monera Kingdom.

Bacteria: As used herein, the terms "bacteria" and "eubacteria" refer to prokaryotic organisms that are differentiable from the Archaea. Similarly, Archaea refers to prokaryotes that are

25 differentiable from eubacteria. Eubacteria and Archaea can be differentiated by various morphological and biochemical criteria. For example, differences in the sequences of ribosomal RNA, RNA polymerase structure, the presence or absence of introns, sensitivity to antibiotics, the presence or absence of peptidoglycans in the cell wall and other cell wall components, branched structures versus a unbranched membrane lipids and the presence / absence of histones and histone-like proteins are used to assign an organism in Eubacteria or in Archaea.

Examples of Eubacteria include Escherichia coli, Thermus thermophilus, Bacillus subtilis and Bacillus stearothermophilus. Examples of Arcaea include Methanococcus jannaschii (Mj), Methanosarcina mazei (Mm), Methanobacterium thermoautotrophicum (Mt), Methanococcus maripaludis, Methanopyrus kandleri, Halobacterium

35 such as Haloferax volcanii and species of Halobacterium NRC-1, Archaeoglobus fulgidus (Af), Pyrococcus furiosus (Pf), Pyrococcus horikoshii (Ph), Pyrobaculum aerophilum, Pyrococcus abyssi, Sulfolobus solfataricus (Ssopusumum (Ss) permixum (Ss) permixum (Ss) permixum (Ssopusumum) , Thermoplasma acidophilum and Thermoplasma volcanium.

Conservative Variant: as used herein, the term "conservative variant" in the context of a translation component, refers to a translation component, for example, a conservative variant O-tRNA or a conservative variant O-RS that functionally is it behaves similarly to a base component to which the conservative variant is similar, for example, an O-tRNA or an O-RS, which has variations in the sequence compared to an O-tRNA or a reference O-RS. For example, an O-RS, or a conservative variant of this O-RS, will aminoacylate a related O-tRNA with a natural amino acid, for example, sulfotyrosine. In

Four. Five In this example, the O-RS and the conservative variant O-RS do not have the same amino acid sequences. The conservative variant can have, for example, a variation, two variations, three variations, four variations or five or more variations in the sequence, provided that the conservative variant is complementary (for example, works with) to the O-tRNA or O- Corresponding related RS.

In some embodiments, a conservative variant O-RS comprises one or more conservative amino acid substitutions compared to the O-RS from which it is derived. In some embodiments, a conservative variant O-RS comprises one or more conservative amino acid substitutions compared to the O-RS from which it is derived, and additionally preserves O-RS biological activity; for example, a conservative variant O-RS that retains at least 10% of the biological activity of the parental O-RS molecule from which it is derived, or as

55 alternative, at least 20%, at least 30% or at least 40%. In some preferred embodiments, the conservative variant O-RS retains at least 50% of the biological activity of the parental O-RS molecule from which it is derived. Conservative amino acid substitutions of a conservative variant O-RS can occur in any domain of the O-RS, including the amino acid binding pocket.

Selection or identification agent: as used herein, the term "selection or identification agent" refers to an agent that, when present, allows the selection / identification of certain components of a population. For example, a selection or identification agent may be, but not limited to, for example, a nutrient, an antibiotic, a wavelength of light, an antibody, an expressed polynucleotide or the like. The selection agent can be varied, for example, by concentration, intensity, etc.

65 In response to: as used herein, the expression "in response to" refers to the process in which an O-tRNA of the invention recognizes a selector codon and mediates the incorporation of the unnatural amino acid, which is coupled to the TRNA, in the growing polypeptide chain.

5 Encode: As used herein, the term "encode" refers to any process by which information in a polymeric macromolecule or sequence chain is used to direct the production of a second molecule or sequence chain that is different. of the first molecule or sequence chain. As used herein, the term is widely used, and may have various applications. In some aspects, the term "encodes" describes the process of semi-conservative DNA replication, where a chain of a double-stranded DNA molecule is used as a template to encode a complementary sister chain newly synthesized by a DNA-dependent DNA polymerase.

In another aspect, the term "encode" refers to any process by which information in a molecule is used to direct the production of a second molecule that has a different chemical nature from the first.

fifteen molecule. For example, a DNA molecule can encode an RNA molecule (for example, by the transcription process incorporating a DNA-dependent RNA polymerase enzyme). In addition, an RNA molecule can encode a polypeptide, as in the translation process. When used to describe the translation process, the term "encode" also extends to the triplet codon that encodes an amino acid. In some aspects, an RNA molecule can encode a DNA molecule, for example, by the reverse transcription process incorporating an RNA-dependent DNA polymerase. In another aspect, a DNA molecule can encode a polypeptide, where it is understood that "coding," as used herein, incorporates both the translation and transcription processes.

Brief description of the figures

25 FIG. 1 provides the chemical structure of the unnatural amino acid sulfothyrosine.

FIG. 2 shows a denaturing PAGE gel stained with Coomassie blue which illustrates the migration of desulfohirudin and sulfohirudin. The size of hirudin cannot be judged by molecular weight patterns due to the atypical load of hirudin.

FIGS. 3A and 3B provide representative graphs of thrombin inhibition with their respective adjusted progress curves superimposed on the unprocessed data points. Enzymatic assays were performed with 50 μM fluorogenic substrate, 40 pM human α-thrombin and 100 pM expressed hirudin in a

35 Tris-HCl saline buffer supplemented with polyethylene glycol 600 and HSA. FIG. 3A shows fluorescence intensity graphs over time without inhibition (control), with desulfohirudin inhibition and with sulfohirudin inhibition. FIG. 3B shows enlargement of desulfohirudin and sulfohirudin graphs for comparison.

FIGS. 4A and 4B illustrate the sulfotyrosine-dependent expression of the Z domain. FIG. 4A provides a denaturing PAGE gel stained with Coomassie blue from the cell lysate purified with Ni-NTA (nitritylotriacetic nickel acid) from cells expressing the Z domain with an amber codon at position 7. Expression only with sulfotyrosine supplemented medium produces the full length Z domain. FIG. 4B provides a linear-positive MALDI-TOF spectrum of positive ions (generated using a THAP matrix) of the lysate

Four. Five Purified cell with Ni-NTA (concentrated and dialyzed against water) showing a peak corresponding to the full length Z domain that contains a single sulfothyrosine and lacks methionine. A peak corresponding to sulfate loss resulting from the conditions of mass spectral analysis is also observed.

FIGS. 5A, 5B and 5C show various MALDI-TOF spectra. FIG. 5A shows a linear-positive MALDI-TOF spectrum of positive ions (generated using a THAP matrix) of pure sulfohirudin showing both the intact sulfohirudin peak [M + H] (7059 Da) and the sulfate loss peak during mass spectral analysis (6979 Da). Note that the small peaks to the right of the main ones are sodium adducts. They occur at additional intervals of 22 Da. FIG. 5B shows a

55 MALDI-TOF spectrum (generated using a synapine matrix) that documents the purity of the sample. To increase the detection of possible impurities, a more rigorous synapine matrix was used, which resulted in the predominance of the peak [M + H-80]. The peak at 13964 Da can be attributed to the dimerization of sulfohirudin. No other impurities were observed. FIG. 5C shows an enlargement of the relevant region that shows the presence of both the intact sulfohirudin peak and [M + H-80]. Intact sulfohirudin is the smallest peak due to the use of the most rigorous synapine matrix. The small peaks to the right of the main ones are sodium adducts.

FIGS. 6A and 6B show various MALDI-TOF spectra. FIGURE 6A shows a MALDI-TOF spectrum (generated using a synapine matrix) of expression means of the unpurified sulfohirudin 65 corresponding to the expression in the absence of sulfothyrosine. Only the truncated hirudin peak was found; no full length protein was observed. FIG. 6B shows a MALDI-TOF spectrum (generated using a

synapine matrix) of unpurified sulfohirudin expression media corresponding to the expression in the presence of sulfotyrosine demonstrating the proportion of truncated sulfohirudin peaks with respect to full length. Due to the more stringent conditions necessary for a good detection of the crude sample mixtures, only the ionized form of sulfohirudin was clearly observed.

5 FIG. 7 provides nucleotide and amino acid sequences.

Detailed description of the invention

Although tyrosine sulfation is a generalized post-translational modification in multicellular eukaryotes (Moore, "The biology and enzymology of protein tyrosine O-sulfation," J Biol Chem 278: 24243-24246 (2003)), its biological functions remain unknown in big measure. This is due in part to the difficulties associated with the synthesis of selectively sulfated proteins. The invention provides selective incorporation of sulfothyrosine into proteins in bacteria by genetic coding of the modified amino acid in response to the nonsense codon.

fifteen Amber, TAG. In addition, it is demonstrated that sulfohirudin, previously inaccessible through recombinant methods, can be expressed directly in E. coli using this strategy. As described herein, kinetic analyzes show an improvement in affinity greater than 10 times against human thrombin by sulfohirudin over desulfohirudin, an observation that offers clinical advantages for sulfohirudin in its use as an anticoagulant (Di Nisio et al ., "Direct thrombin inhibitors," N Engl J Med 353: 10281040 (2005)). This general strategy for the biosynthesis of sulfated proteins facilitates the study and additional application of the emergent post-translational modification, the sulphation of tyrosine.

As a general method for protein site specific sulfation, the present description describes the development of an orthogonal tRNA / aminoacyl-tRNA synthetase (aaRS) pair that allows efficient and selective incorporation

25 of sulfothyrosine in eukaryotic proteins such as E. coli in response to the amber senseless codon. Using this unique suppressor tRNA / aaRS pair, the native sulphated form of hirudin is directly expressed and shown to have an affinity 10 times greater for human thrombin than for desulfohirudin, according to previous literature publications (Stone and Hofsteenge , "Kinetics of the inhibition of thrombin by hirudin," Biochemistry 25: 4622-4628 (1986)).

The present specification provides orthogonal tRNA / aminoacyl-tRNA synthetase pairs that allow selective in vivo introduction of sulfothyrosine (see FIG. 1) into proteins in E. coli in response to a selector codon, for example, the termination codon amber TAG. The invention provides new orthogonal aminoacyl-tRNA synthetase (O-RS) polypeptides, which specifically load an orthogonal tRNA (O-tRNA) related to the

35 unnatural amino acid sulfothyrosine.

In some aspects, to demonstrate (but not limit) the present invention, the description herein demonstrates that the rest of the unnatural amino acid can be incorporated into various model proteins. It is not intended to limit the incorporation of the unnatural amino acid to any particular protein. From the present description, it will be obvious that the incorporation of the unnatural amino acid sulfothyrosine into proteins of particular interest is advantageous for a wide variety of purposes.

The present description describes the development of new orthogonal tRNA / aminoacyl-tRNA synthetase pairs that function in eubacteria to specifically incorporate an unnatural sulfotyrosine amino acid into the site

Four. Five (provided in FIG. 1) in response to selector codons. In summary, the invention provides novel tyrosyl-tRNA synthetase mutants of Methanococcus janaschii as defined in the claims, which selectively load a suppressor tRNA with the non-natural amino acid sulfothyrosine into E. coli host cells.

These developed tRNA synthetase pairs can be used to specifically incorporate in the site the unnatural amino acid sulfothyrosine into a protein. The incorporation of the unnatural amino acid into the protein can be programmed to occur at any desired position by genetically engineered modification of the polynucleotide encoding the protein of interest to contain a selector codon that signals the incorporation of the unnatural amino acid.

55 The invention described herein provides orthogonal pairs for the coding and genetic incorporation of the unnatural amino acid sulfothyrosine into proteins in a eubacterium, for example, an E. coli cell, in which orthogonal components do not cross-react with components of AND. Endogenous coli from the host cell's translational machinery, but recognize the desired unnatural amino acid and incorporate it into proteins in response to a selector codon (for example, a nonsense amber codon, TAG). The orthogonal components provided by the invention include orthogonal aminoacyl-tRNA synthetases derived from Methanococcus janaschii tRNA syntheses and the amber tyrosyl ARNtCUA suppressor, a mutant that functions as an orthogonal pair in a eubacteria host cell.

The invention provides compositions and methods, as defined in the claims, for identifying and

65 produce additional pairs of orthogonal tRNA-aminoacyl-tRNA synthetase, for example O-tRNA / O-RS pairs that can be used to incorporate sulfothyrosine into proteins. An O-tRNA / O-RS pair of the invention can mediate the incorporation of sulfothyrosine into a protein that is encoded by a polynucleotide, in which the polynucleotide comprises a selector codon that is recognized by the O-tRNA. The anticodon loop of the O-tRNA recognizes the selector codon in an mRNA and incorporates the unnatural amino acid at this site into the polypeptide. Generally, an orthogonal aminoacyl-tRNA synthetase of the invention aminoacyl (or charge) preferably its O-tRNA only with a

5 specific unnatural amino acid.

ARNt TECHNOLOGY / AMINOACIL-ARNt ORTOGONAL SYNTHEASE

An understanding of the new compositions and methods of the present invention requires an understanding of

10 the activities associated with orthogonal tRNA pairs and orthogonal aminoacyl-tRNA synthetase. To add additional unnatural amino acids to the genetic code, new orthogonal pairs are required that comprise an aminoacyl-tRNA synthetase and a suitable tRNA that can function effectively in the host's translational machinery, but are "orthogonal" for the translation system in question. , which means that they work independently of synthetases and tRNAs endogenous to the translation system. characteristics

fifteen desired of the orthogonal pair include tRNA that decodes or recognizes only a specific codon, for example, a selector codon, which is not decoded by any endogenous tRNA, and aminoacyl-tRNA synthetases that aminoacylate (or "load") preferentially their related tRNA only with a specific unnatural amino acid. In addition, the O-tRNA is not normally aminoacylated (or is poorly aminoacylated, that is, charged) by endogenous synthetases. For example, in an E. host system. coli, an orthogonal pair will include an aminoacyl-tRNA synthetase that does not react in

twenty crossed with none of the endogenous tRNAs, for example, in which there are 40 in E. coli, and an orthogonal tRNA that is not aminoacylated by any of the endogenous synthetases, for example, of which there are 21 in E. coli.

The general principles of orthogonal translation systems that are suitable for making proteins comprising one or more natural amino acids are known in the art, as well as in general methods for the production of orthogonal translation systems. For example, see International Publication Numbers WO 2002/086075, entitled "METHODS AND COMPOSI-TION FOR THE PRODUCTION OF ORTHOGONAL tRNA-AMINOACYL-tRNA SYNTHETASE PAIRS"; WO 2002/085923, entitled "IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS"; WO 2004/094593, entitled "EXPANDING THE EUKARYOTIC GENETIC CODE"; WO 2005/019415, filed July 7, 2004; WO 2005/007870, filed July 7, 2004; WO 2005/007624, filed July 7, 2004; WO 2006/110182, filed on October 27, 2005, entitled “ORTHOGONAL TRANSLATION COMPONENTS FOR THE VIVO INCORPORATION OF UNNATURAL AMINO AIDS” and WO 2007/103490, filed on March 7, 2007, titled “SYSTEMS FOR THE EXPRESSION OF ORTHOGONAL TRANSLATION COMPONENTS IN EUBACTERIAL HOST CELLS ”. For a debate on orthogonal translation systems that incorporate unnatural amino acids and methods for their production and use,

35 see also Wang and Schultz "Expanding the Genetic Code," Angewandte Chemie Int. Ed., 44 (1): 34-66 (2005), Xie and Schultz, "An Expanding Genetic Code," Methods 36 (3): 227- 238 (2005); Xie and Schultz, "Adding Amino Acids to the Genetic Repertoire," Curr. Opinion in Chemical Biology 9 (6): 548-554 (2005); and Wang et al., "Expanding the Genetic Code," Annu. Rev. Biophys. Biomol Struct., 35: 225-249 (2006).

40 Orthogonal translation systems

Orthogonal translation systems generally comprise cells (which can be prokaryotic cells such as E. coli) that include an orthogonal tRNA (O-tRNA), an orthogonal aminoacyl tRNA synthetase (O-RS), and an unnatural amino acid, in the that the O-RS aminoacylates the O-tRNA with the unnatural amino acid. An orthogonal pair of the invention may include an O-tRNA, for example, a suppressor tRNA, a reading phase shift tRNA,

or similar, or a related O-RS. The orthogonal systems of the invention defined in the claims normally comprise O-tRNA / O-RS pairs, in the context of a host cell or without the host cell. In addition to multicomponent systems, the invention also provides new individual components, for example, new orthogonal aminoacyl-tRNA synthetase polypeptides (eg, SEQ ID Nos: 4, 6, 8 or 10), and

fifty polynucleotides encoding these polypeptides (eg, SEQ ID Nos: 5, 7, 9 or 11).

In general, when an orthogonal pair recognizes a selector codon and loads an amino acid in response to the selector codon, the orthogonal pair is said to "suppress" the selector codon. That is, a selector codon that is not recognized by the endogenous machinery (for example, of the cell) of the translation system, is not usually

55 loaded, which results in the blockage of the production of a polypeptide that would otherwise be transcribed from the nucleic acid. In an orthogonal pair system, the O-RS aminoacylates the O-tRNA with a specific unnatural amino acid. The loaded O-tRNA recognizes the selector codon and suppresses the translational lock produced by the selector codon.

60 In some aspects, an O-tRNA of the invention recognizes a selector codon and may include a suppression efficiency of at least about, for example, 45%, 50%, 60%, 75%, 80% or 90% or more in presence of a related synthetase in response to a selector codon compared to the suppression efficiency of an O-tRNA that comprises or is encoded by a polynucleotide sequence as indicated in the sequence list herein.

65 In some embodiments, the suppression efficiency of the O-RS and the O-tRNA as a whole may be approximately, for example, 5 times, 10 times, 15 times, 20 times or 25 times or higher than the suppression efficiency. of the O-tRNA that lacks the O-RS. In some aspects, the suppression efficiency of the O-RS and the O-tRNA as a whole may be at least about, for example, 35%, 40%, 45%, 50%, 60%, 75%, 80

5 % or 90% or more of the suppression efficiency of an orthogonal synthetase pair as indicated in the sequence list herein.

The host cell uses the O-tRNA / O-RS pair that incorporates the unnatural amino acid into a growing polypeptide chain, for example, by a nucleic acid comprising a polynucleotide encoding a polypeptide of interest, in which the polynucleotide comprises a selector codon that is recognized by the O-tRNA. In certain preferred aspects, the cell may include one or more additional O-tRNA / O-RS pairs, in which the additional O-tRNA is charged by the additional O-RS with a different non-natural amino acid. For example, one of the tRNAs can recognize a four base codon and the other O-tRNA can recognize a termination codon. Alternatively, different termination codons can be used in the same coding nucleic acid

fifteen multiple or codons of four different different bases.

As can be seen, in some embodiments, there are multiple O-tRNA / O-RS pairs in a cell or other translation system, which allow the incorporation of more than one unnatural amino acid into a polypeptide. For example, the cell may additionally include an additional different O-tRNA / O-RS pair and a second unnatural amino acid, in which this O-tRNA recognizes a second selector codon and this additional O-RS preferably aminoacylates the O-tRNA with the second unnatural amino acid. For example, a cell that includes an O-tRNA / O-RS pair (in which the O-tRNA recognizes, for example, an amber selector codon), may additionally comprise a second orthogonal pair, in which second O-tRNA recognizes a different selector codon, for example, an opal codon, a four base codon or the like. Desirably, the different orthogonal pairs derive from different sources, which

25 They can facilitate the recognition of different selector codons.

In certain embodiments, the systems comprise a cell, such as an E. coli cell that includes an orthogonal tRNA (O-tRNA), an orthogonal aminoacyl-tRNA synthetase (O-RS), an unnatural amino acid and a nucleic acid comprising a polynucleotide encoding a polypeptide of interest, in which the polynucleotide comprises the selector codon that is recognized by the O-tRNA. The translation system can also be an acellular system, for example, any of several commercially available "in vitro" transcription / translation systems in combination with an O-tRNA / O-RS pair and an unnatural amino acid as described. in the present document.

35 The O-tRNA and / or the O-RS can be of natural origin or can be derived, for example, by mutation of a tRNA and / or a naturally occurring RS, for example, generating tRNA libraries and / or RS libraries , from any of several organisms and / or using any of several mutation strategies available. For example, a strategy for producing an orthogonal tRNA / aminoacyl-tRNA synthetase pair involves importing a heterologous tRNA / synthetase pair (to the host cell) from, for example, a source other than the host cell, or multiple sources, in the host cell. The properties of the heterologous candidate synthetase include, for example, that it does not load any tRNA into the host cell, and the properties of the heterologous tRNA candidate include, for example, that it is not aminoacylated by any host cell synthetase. In addition, the heterologous tRNA is orthogonal with respect to all synthetases of the host cell. A second strategy for generating an orthogonal pair involves generating mutant libraries from which an O-tRNA is identified and / or selected.

Four. Five an O-RS. These strategies can also be combined.

Orthogonal tRNA (O-tRNA)

An orthogonal tRNA (O-tRNA) of the invention desirably mediates the incorporation of an unnatural amino acid into a protein that is encoded by a polynucleotide comprising a selector codon that is recognized by the O-tRNA, for example, in vivo or in. vitro. In certain embodiments, an O-tRNA of the invention may include a suppression efficiency of at least about, for example, 45%, 50%, 60%, 75%, 80% or 90% or more. in the presence of a related synthetase in response to a selector codon compared to an O-tRNA that comprises or is encoded by a polynucleotide sequence as indicated in the O-tRNA sequences

55 in the sequence list of this document.

The suppression efficiency can be determined by any of several assays known in the art. For example, an indicator assay with β-galactosidase can be used, for example, a derivatized lacZ plasmid (in which the construct has a selector codon in the lacZ nucleic acid sequence) is introduced into cells of an appropriate organism (for example , an organism in which orthogonal components may be used) together with plasmid comprising an O-tRNA of the invention. A related synthetase may also be introduced (such as a polypeptide or a polynucleotide encoding the related synthetase when expressed). Cells are cultured in media at a desired density, for example, at an OD600 of about 0.5, and βgalactosidase assays are performed, for example, using the BetaFluor ™ β-Galactosidase Assay Kit (Novagen). The percentage of deletion can be calculated as the percentage of activity for a sample with respect to a comparable control, for example, the value observed from the derivatized lacZ construction, in which the construction has a

corresponding sense codon in the desired position instead of a selector codon.

In the sequence list of this document, for example, see FIG. 7 and SEQ ID NO: 1 examples of O-tRNA of the invention are indicated. The description of this document also provides guidance

5 for the design of equivalent additional O-tRNA species. In an RNA molecule, such as an O-RS mRNA, or O-tRNA molecule, Thymine (T) is replaced with Uracil (U) with respect to a given sequence (or vice versa for a coding DNA), or its complement. Additional modifications to the bases may also be present to generate largely functionally equivalent molecules.

The invention also includes conservative variations of O-tRNAs corresponding to particular O-tRNAs herein. For example, conservative variations of O-tRNA include those molecules that function as particular O-tRNAs, for example, as in the sequence list of this document and that retain the L-shaped structure of tRNA due to appropriate self-complementarity , but which do not have a sequence identical to those of, for example, the sequence list of FIG. 7, and desirably, are different from those

fifteen wild type tRNA molecules.

The composition comprises an O-tRNA that additionally includes an orthogonal aminoacyl-tRNA synthetase (O-RS) as defined in the claims, wherein the O-RS preferably aminoacylates the O-tRNA with an unnatural amino acid. In certain embodiments, a composition that includes an O-tRNA may additionally include a translation system (eg, in vitro or in vivo). In the cell there can also be a nucleic acid comprising a polynucleotide encoding a polypeptide of interest, in which the polynucleotide comprises a selector codon recognized by the O-tRNA, or a combination of one or more of these.

Production methods of an orthogonal tRNA (O-tRNA) are also an aspect of the present invention. One

25 TRNA produced by the method is used in the compositions and methods of the invention. In certain embodiments of the present description, the O-tRNAs can be produced by generating a library of mutants. The library of mutant tRNAs can be generated using various mutagenesis techniques known in the field. For example, mutant tRNAs can be generated by site-specific mutations, random point mutations, homologous recombination, DNA transposition or other methods of recursive mutagenesis, chimeric construction or any combination thereof, for example, from the SEC O-tRNA. ID Nº: 1.

Additional mutations may be introduced in a specific position (or positions), for example, in a non-conservative position (or positions), or in a conservative position, in a random position (or positions), or a combination of both in a loop desired as a region of an tRNA, for example, an anticodon loop, the stem

35 acceptor, arm or D loop, variable loop, arm or TPC loop, other regions of the tRNA molecule, or a combination thereof. Typically, mutations in an tRNA include mutating the anticodon loop of each member of the library of mutant tRNAs to allow recognition of a selector codon. The method may include additionally adding additional sequences to the O-tRNA. Normally, an O-tRNA has an orthogonality improvement for a desired organism compared to the starting material, for example, the plurality of tRNA sequences, while retaining its affinity towards a desired RS.

The methods include optionally analyzing the similarity (and / or deduced homology) of sequence of tRNAs and / or aminoacyl-tRNA synthetases to determine potential candidates for an O-tRNA, O-RS and / or pairs thereof, which appear to be orthogonal for a specific organism. Computer programs known in the art and described

Four. Five Here, BLAST can be used for analysis, for example, and compilation programs can be used. In one example, to select possible orthogonal translation components for use in E. coli, a synthetase and / or an tRNA that does not show sequence similarity to eubacterial organisms is selected.

Normally, an O-tRNA is obtained by subjecting, for example, negative selection, a population of cells of a first species, in which the cells comprise a member of the plurality of possible O-tRNAs. Negative selection eliminates cells that comprise a member of the library of possible O-tRNAs that is aminoacylated by an aminoacyl-tRNA synthetase (RS) that is endogenous to the cell. This provides a set of tRNAs that are orthogonal to the cell of the first species.

55 In certain embodiments, in the negative selection, one or more selector codons are introduced into a polynucleotide encoding a negative selection marker, for example, an enzyme that confers antibiotic resistance, for example, β-lactamase, an enzyme that confers a detectable product, for example, βgalactosidase, chloramphenicol acetyltransferase (CAT), for example, a toxic product, such as barnase, in a non-essential position (for example, which still produces a functional barnase), etc. Detection / selection is optionally performed by culturing the cell population in the presence of a selective agent (eg, an antibiotic, such as ampicillin). In one embodiment, the concentration of the selection agent is modified.

For example, to measure the activity of tRNA suppressors, a selection system is used that is based on the

65 in vivo deletion of selector codon, for example, nonsense (eg, detection) or phase shift mutations introduced into a polynucleotide encoding a negative selection marker, for example, a gene for β-lactamase (bla). For example, polynucleotide variants, for example bla variants, are constructed with a selector codon at a certain position (for example, A184). Cells, for example, bacteria, are transformed with these polynucleotides. In the case of an orthogonal tRNA, which cannot be efficiently loaded by endogenous E. coli synthetases, antibiotic resistance, for example, ampicillin resistance,

5 it must be approximately or less than that for which a bacterium was transformed without a plasmid. If the tRNA is not orthogonal, or if a heterologous synthetase capable of loading the tRNA is coexpressed in the system, a higher level of antibiotic is observed, for example, ampicillin resistance. Cells are selected, for example, bacteria, which cannot be cultured on LB agar plates with antibiotic concentrations approximately equal to transformed cells without plasmids.

In the case of a toxic product (for example, ribonuclease or barnase), when a member of the plurality of possible tRNAs is aminoacylated by an endogenous host, for example, Escherichia coli synthetases (ie, it is not orthogonal to the host, by example, Escherichia coli synthetases) the selector codon is suppressed and the toxic polynucleotide product produced leads to cell death. Cells that house orthogonal tRNAs or

fifteen Non-functional tRNAs survive.

In one embodiment, the set of tRNAs that are orthogonal to a desired organism are then subjected to a positive selection in which a selector codon is placed on a positive selection marker, for example, encoded by a drug resistance gene, such as a β-lactamase gene. The positive selection is made in a cell that comprises a polynucleotide that encodes or that comprises a member of the set of tRNAs that are orthogonal to the cell, a polynucleotide that encodes a positive selection marker and a polynucleotide that encodes a related RS. In certain embodiments, the second population of cells comprises cells that were not removed by negative selection. The polynucleotides are expressed in the cell and the cell is cultured in the presence of a selection agent, for example, ampicillin. Then the tRNAs are selected for their ability to

25 aminoacylate by co-expressed related synthetase and to insert an amino acid in response to this selector codon. Normally, these cells show potentiation in terms of suppression efficacy compared to cells that host non-functional tRNA (s), or tRNAs that cannot be effectively recognized by the synthetase of interest. Cells that house non-functional tRNAs or tRNAs that are not effectively recognized by the synthetase of interest, are sensitive to the antibiotic. Therefore, tRNAs that: (i) are not substrates for endogenous host synthetases, for example, from Escherichia coli; (ii) they can be aminoacylated by the synthetase of interest; and (iii) they are functional in translation, they survive both selections.

Therefore, the same marker can be a positive or negative marker, depending on the context in which it is explored. That is, the same marker is a positive marker if it is scanned for, but it is a marker.

35 negative if scanned against.

The rigor of the selection, for example, the positive selection, the negative selection or both the positive and the negative selection, in the methods described above, optionally includes modifying the rigor of the selection. For example, since barnase is an extremely toxic protein, the stringency of the negative selection can be controlled by introducing different numbers of selector codons into the barnase gene and / or using an inducible promoter. In another example, the concentration of the selection or identification agent is modified (eg, ampicillin concentration). In some aspects, the rigor is modified because the desired activity may be low during the first rounds. Therefore, less stringent selection criteria are applied in the first rounds and more stringent criteria are applied in subsequent rounds of selection. In certain

Four. Five embodiments, the negative selection, the positive selection or both the positive and the negative selection can be repeated multiple times. Multiple different negative selection markers, positive selection markers or both positive and negative selection markers can be used. In certain embodiments, the positive and negative selection marker may be the same.

Other types of selection / identification can be used in the methods of the invention to produce orthogonal translation components, for example, an O-tRNA, an O-RS and an O-tRNA / O-RS pair that loads an unnatural amino acid into response to a codon selector. For example, the negative selection marker, the positive selection marker or both the positive and negative selection marker may include a marker that produces fluorescence or catalyzes a luminescent reaction in the presence of a suitable reagent. In another embodiment, you can

55 a product of the marker can be detected by separation of fluorescence activated cells (FACS) or by luminescence. Optionally, the marker includes an affinity based identification marker. See also Francisco, JA, et al., (1993) Production and fluorescence-activated cell sorting of Escherichia coli expressing a functional antibody fragment on the external surface. Proc Natl Acad Sci US A. 90: 10444-8.

Additional methods for producing a recombinant orthogonal tRNA can be found, for example, in International Application Publications WO 2002/086075, entitled "METHODS AND COMPOSITIONS FOR THE PRODUCTION OF ORTHOG-ONAL tRNA AMINOACYL-tRNA SYNTHETASE PAIRS"; WO 2004/094593, entitled "EXPANDING THE EUKARYOTIC GENETIC CODE" and WO 2005/019415, filed July 7, 7, 2004. See also Forster et al., (2003) Programming peptidomimetic synthetases by translating genetic codes designed by

65 novo PNAS 100 (11): 6353-6357; and, Feng et al., (2003), Expanding tRNA recognition of a tRNA synthetase by a single amino acid change, PNAS 100 (10): 5676-5681.

Orthogonal Aminoacyl-tRNA synthetase (O-RS)

An O-RS of the invention is defined in the claims and preferably an amino acid O-tRNA with an unnatural amino acid, in vitro or in vivo. An O-RS of the invention can be provided to the translation system, for example, a cell, by a polypeptide that includes an O-RS and / or a polynucleotide encoding an O-RS

or a part of it. For example, an illustrative O-RS comprises an amino acid sequence as set forth in SEQ ID Nos: 4, 6, 8 or 10 or a conservative variation thereof. In another example, an O-RS, or a part thereof, is encoded by a polynucleotide sequence encoding a sequence comprising amino acids in the sequence list or the examples herein, or a polynucleotide sequence.

10 complementary to it. See, for example, the polynucleotide of SEQ ID Nos: 5, 7, 9 or 11.

The methods as defined in the claims may comprise identifying an orthogonal aminoacyl-tRNA synthetase (O-RS), for example, an O-RS, for use with an O-tRNA. For example, one method includes subjecting, for example, positive selection, a population of cells of a first species, in which cells 15 individually comprise: 1) a member of a plurality of aminoacyl-tRNA synthetases (RS) ( for example, the plurality of RS may include mutant RS, RS derived from a species other than the first species or both mutant RS and RS derived from a species other than the first species); 2) orthogonal tRNA (O-tRNA) (for example of one or more species); and 3) a polynucleotide that encodes a selection marker (eg, positive) and comprises at least one selector codon. Cells that show an increase in suppression efficacy are selected or identified compared to cells lacking or with a reduced amount of the member of the plurality of RS. The suppression efficiency can be measured by techniques known in the field and described herein. Cells that have an increase in suppression efficiency comprise an active RS that aminoacylates the O-tRNA. A level of aminoacylation (in vitro or in vivo) by the active RS of a first set of tRNA of the first species is compared with the level of aminoacylation (in vitro or in vivo) by the active RS of a second

25 tRNA set of the second species. The level of aminoacylation can be determined by a detectable substance (for example, a labeled unnatural amino acid). Normally, the active RS is selected that effectively aminoacylates the second set of tRNAs compared to the first set of tRNAs, thereby providing an effective orthogonal tRNA aminoacyl synthetase (optimized) for use with the O-tRNA. An O-RS, identified by the method, is also a feature of the invention.

30 Any of several assays can be used to determine aminoacylation. These tests can be performed in vitro or in vivo. For example, in vitro aminoacylation assays are described, for example, in Hoben and Soll (1985) Methods Enzymol. 113: 55-59. Aminoacylation can also be determined using an indicator together with the orthogonal translation components and detecting the indicator in a cell that expresses a

35 polynucleotide comprising at least one selector codon that encodes a protein. See also, WO 2002/085923, entitled "IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS"; and WO 2004/094593, entitled "EXPANDING THE EUKARYOTIC GENETIC CODE".

The identified O-RS can be further manipulated to alter the substrate specificity of the synthetase, from

40 such that only a desired unnatural amino acid, but none of the 20 common amino acids, is loaded into the O-tRNA. Methods for generating an orthogonal aminoacyl-tRNA synthetase with a substrate specificity for a natural amino acid include mutating the synthetase, for example, at the active site in the synthetase, at the site of the synthetase editing mechanism, at different sites by combining different domains of synthetases, or the like, and apply a selection process. A strategy based on the combination of a selection is used

Four. Five positive followed by a negative selection. In positive selection, deletion of the selector codon introduced at one or more or nonessential positions of a positive marker allows cells to survive under positive selection pressure. In the presence of both natural and non-natural amino acids, survivors thus encode active synthetases that load orthogonal suppressor tRNA with a natural or unnatural amino acid. In the negative selection, the deletion of a selector codon introduced in one position or more non-essential positions of a

fifty Negative marker eliminates synthetases with natural amino acid specificities. Survivors of the negative and positive selection encode synthetases that aminoacylate (charge) orthogonal suppressor tRNA only with unnatural amino acids. Then, these synthetases can undergo additional mutagenesis, for example, DNA transposition or other recursive mutagenesis methods.

55 A library of mutant O-RSs can be generated using various mutagenesis techniques known in the art. For example, mutant RSs can be generated by site specific mutations, random point mutations, homologous recombination, DNA transposition or other methods of recursive mutagenesis, chimeric construction or any combination thereof. For example, a library of mutant RSs can be produced from two or more, for example, different, smaller, less diverse "sub-libraries." As well

60 chimeric RS libraries are described. It should be noted that libraries of tRNA synthetases of various organisms (for example, microorganisms, such as eubacteria or archaebacteria) such as libraries comprising natural diversity (see, for example, U.S. Patent No. 6,238,884 to Short et al. ; United States Patent No. 5,756,316 to Schallenberger et al .; United States Patent No. 5,783,431 to Petersen et al .; United States Patent No. 5,824,485 to Thompson et al .; U.S. Patent No.

65 5,958,672 to Short et al.) Are optionally constructed and explored for orthogonal pairs.

Once the synthetases have undergone the positive and negative selection / identification strategy, these synthetases can then undergo additional mutagenesis. For example, a nucleic acid encoding the O-RS can be isolated; a set of polynucleotides encoding the mutated O-RSs (for example, by random mutagenesis, site-specific mutagenesis, recombination or any combination thereof) can be generated at

5 starting from nucleic acid; and these individual stages or a combination of these stages can be repeated until a mutated O-RS is obtained that preferably aminoacylates the O-tRNA with the unnatural amino acid. In some aspects of the invention, the steps can be performed multiple times, for example, at least twice.

In the methods of the invention, additional levels of selection / identification stringency can also be used to produce O-tRNA, O-RS, or pairs thereof. The rigor of the selection or identification may vary in one or both stages of the method of producing an O-RS. This could include, for example, varying the amount of the selection / identification agent used, etc. Additional rounds of positive and / or negative selections can also be used. The selection or identification may also comprise one or more of a change in amino acid permeability, a change in translation efficiency, a change in the

fifteen translation fidelity, etc. Typically, said one or more changes are based on a mutation in one or more genes in an organism in which an orthogonal tRNA tRNA synthetase pair is used to produce a protein.

Additional general details for the production of O-RS and the modification of the specificity of synthetase by the substrate can be found in Internal Publication Number WO 2002/086075, entitled “METHODS AND COMPOSITIONS FOR THE PRODUCTION OF ORTHOGONAL tRNA AMINOACYL-tRNA SYNTHETASE PAIRS "; and in WO 2004/094593, entitled "EXPANDING THE EUKARYOTIC GENETIC CODE". See also Wang and Schultz "Expanding the Genetic Code," Ange-wandte Chemie Int. Ed., 44 (1): 34-66 (2005).

SOURCE ORGANISMS AND HOSPEDADORES

25 The orthogonal translation components (O-tRNA and O-RS) of the invention defined in the claims may be derived from any organism (or a combination of organisms) for use in a host translation system of any other species, with the warning that the O-tRNA / O-RS components and the host system act in an orthogonal manner. It is not necessary that the O-tRNA and the O-RS of an orthogonal pair derive from the same organism. In some aspects, orthogonal components are derived from Archaea genes (i.e., archaebacteria) for use in a eubacterial host system.

For example, the orthogonal O-tRNA may be derived from an Archae organism, for example an archaeobacterium, such as Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium such as Haloferax volcanii and 35 species of Halobacterium NRC-1, Archaeoglobus pyrococcus pycoccususccususcumuscuscuscususus , Aeuropyrum pernix, Methanococcus maripaludis, Methanopyrus kandleri, Methanosarcina mazei (Mm), Pyrobaculum aerophilum, Pyrococcus abyssi, Sulfolobus solfataricus (Ss), Sulfolobus tokodaii, Thermoplasma acidophilum, Thermoplasma volcanium, or the like, or a eubacterium, such as Escherichia coli, Thermus therinophilus, Bacillus subtilis, Bacillus stearothermphilus, or the like, while orthogonal O-RS may derive from an organism or combination of organisms, for example, an archaebacteria, such as Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium such as Haloferax volcanii and Halobacterium NRC-1 species, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, Methanococcus maripaludis, Methanopyrus kandleri, Methanosarcina mazei, Pyrobaculum aerophilum, Pyrococcus abyssi, Sulfolomamous, Sulfolobaum, Sulfolobaum, Sulfolobaum, Sulfolobaum, Sulfolobaum, Sulfolobaum, Solifama, Thermoema, Volcano, Solomatus, Volcano coli, Thermus themiophilus, Bacillus subtilis, Bacillus stearothemiphilus, or the like. In one embodiment, eukaryotic sources can also be used, for example, plants, algae, protists, fungi, yeasts, animals (eg, mammals, insects, arthropods, etc.), or the like, as sources of O-tRNA and O- RS.

The individual components of an O-tRNA / O-RS pair can be derived from the same organism or from different organisms. In one embodiment, the O-tRNA / O-RS pair is derived from the same organism. Alternatively, the O-tRNA and the O-RS of the O-tRNA / O-RS pair are from different organisms.

The O-tRNA, the O-RS or the O-tRNA / O-RS pair can be selected or identified in vivo or in vitro and / or used in a cell, for example, a eubacterial cell, to produce a polypeptide with a natural amino acid The cell of

55 Eubacteria used is not limited, for example, Escherichia coli, Thermus thermophilus, Bacillus subtilis, Bacillus stearothermphilus, or the like. Compositions of eubacterial cells comprising translation components of the invention are also a feature of the invention.

See also International Application Publication Number WO 2004/094593, entitled "EXPANDING THE EUKARYOTIC GENETIC CODE", filed on April 16, 2004, to identify O-tRNA and / or O-RS in one species for use in another species .

Although orthogonal translation systems (for example, comprising an O-RS, an O-tRNA and an unnatural amino acid) can use cultured host cells to produce proteins that have non-natural amino acids, it is not intended that a translation system Orthogonal of the invention requires a viable, intact host cell. For example, an orthogonal translation system can use a cellular system in the presence of a

cell extract In fact, the use of acellular transcription / translation systems in vitro for protein production is a well established technique. The adaptation of these in vitro systems to produce proteins that have unnatural amino acids using orthogonal translation system components described herein is well within the scope of the invention.

5 SELECTOR CODONS

The selector codons of the invention expand the reading phase of the genetic codon of the protein biosynthetic machinery. For example, a selector codon includes, for example, a single three-base codon, a non-sense codon, such as a termination codon, for example, an amber codon (UAG), or an opal codon (UGA), a unnatural codon, at least one four base codon, a rare codon, or the like. Various selector codons can be introduced into a desired gene, for example, one or more, two or more, more than three, etc. Using different selector codons, multiple orthogonal pairs of tRNA synthetase can be used to allow simultaneous site specificity incorporation of multiple unnatural amino acids, for example, including at least one

fifteen unnatural amino acid, using these different selector codons.

In one embodiment, the methods may involve the use of a selector codon that is a termination codon for incorporation of an unnatural amino acid in vivo into a cell. For example, an O-tRNA is produced that recognizes the termination codon and is aminoacylated by an O-RS with an unnatural amino acid. This O-tRNA is not recognized by naturally occurring host aminoacyl-tRNA synthetases. Conventional directed site mutagenesis can also be used to introduce the termination codon at the site of interest into a polynucleotide encoding a polynucleotide of interest. See, for example, Sayers et al. (1988), 5 ', 3' Exonuclease in phosphorothioate-based oligonucleotide-directed mutagenesis. Nucleic Acids Res, 791-802. When the O-RS, O-tRNA and the nucleic acid encoding a polypeptide of interest are combined, for example, in vivo, the amino acid does not

25 Natural is incorporated in response to the termination codon to give a polypeptide containing the unnatural amino acid at the specified position. In one embodiment of the invention, the termination codon used as a selector codon is an amber codon, UAG, and / or can be an opal codon, UGA. In one example, a genetic code in which both UAG and UGA are used as a selector codon can encode 22 amino acids while retaining the UAA nonsense codon, which is the most abundant termination signal.

30 The incorporation of unnatural amino acids in vivo can be performed without significant disturbance of the host cell. For example, in non-eukaryotic cells, such as Escherichia coli, how the suppression efficacy for the UAG codon depends on the competitiveness between the O-tRNA, for example, the amber suppressor tRNA, and the release factor 1 (RF1) ( which binds to the UAG codon and initiates the release of the ribosome growth peptide), the

35 Suppression efficiency can be modulated, for example, by increasing the level of O-tRNA expression, for example, the suppressor tRNA, or by using a strain deficient in RF1. In eukaryotic cells, since the suppression efficiency of a UAG codon depends on the competitiveness between the O-tRNA, for example, the amber suppressor tRNA, and the eukaryotic release factor (for example, eRF) (which binds to a termination codon and initiates the release of the ribosome growth peptide), suppression efficiency can be modulated, for example, by increasing the level of expression

40 of O-tRNA, for example, the suppressor tRNA. In addition, additional compounds may also be present, for example, reducing agents such as dithiothreithiol (DTT).

Unnatural amino acids can also be encoded by rare codons. For example, when the concentration of arginine in a region of protein synthesis in vitro is reduced, the rare AGG arginine codon has been shown to be effective for the insertion of Ala by a synthetic tRNA acylated with alanine. See, for example, Ma et al., Biochemistry, 32: 7939 (1993). In this case, the synthetic tRNA competes with the naturally occurring tRNA, which exists as a minor species in Escherichia coli. In addition, some organisms do not use all triplet codons. An unallocated AGA codon in Micrococcus luteus has been used for amino acid insertion in an in vitro transcription / translation extract. See, for example, Kowal and Oliver, Nucl. Acid Res., 25: 4685 (1997).

fifty Components of the invention can be generated to use these rare codons in vivo.

Selector codons may also comprise extended codons, for example, codons of four or more bases, such as, codons of four, five, six or more bases. Examples of four base codons include, for example, AGGA, CUAG, UAGA, CCCU, and the like. Examples of five base codons include, for example, AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC and the like. The methods of the invention may include using extended codons based on suppression of reading phase shift. Codons of four or more bases, for example, one or multiple unnatural amino acids, can be inserted into the same protein. In other embodiments, the anticodon loops can decode, for example, at least one codon of four bases, at least one codon of five bases or at least one codon of six bases or more. Since there are 256 codons of four

60 possible bases, multiple unnatural amino acids can be encoded in the same cell using a codon of four or more bases. See also, Anderson et al., (2002) "Exploring the Limits of Codon and Anticodon Size," Chemistry and Biology 9: 237-244; and, Magliery, (2001) “Expanding the Genetic Code: Selection of Efficient Suppressors of Four-base Codons and Identification of“ Shifty ”Four-base Codons with a Library Approach in Escherichia coli,” J. Mol. Biol., 307: 755-769.

For example, four-base codons have been used to incorporate unnatural amino acids into proteins using in vitro biosynthetic methods. See, for example, Ma et al., (1993) Biochemistry 32: 7939; and Hohsaka et al., (1999) J. Am. Chem. Soc., 121: 34. CGGG and AGGU were used to simultaneously incorporate 2-naphthylalanine and a lysine NBD derivative in streptavidin in vitro with two chemically acylated 5-phase reading displacement tRNA suppressors. See, for example, Hohsaka et al., (1999) J. Am. Chem. Soc., 121: 12194. In an in vivo study, Moore et al. examined the ability of tRNA derivatives with NCUA anticodons to suppress UAGN codons (N can be U, A, G or C), and observed that the UAGA quadruplet could be decoded by an tRNA with an UCUA anticodon with an efficiency of 13 to 26% with little decoding in phase 0 or -1. See Moore et al., (2000) J. Mol. Biol., 298: 195. In one embodiment, extended codons based on rare codons or nonsense codons can be used in the invention, which can reduce complete nonsense reading and suppression of reading phase shift at other unwanted sites. In various orthogonal systems, four base codons have been used. See, for example, WO 2005/019415; WO 2005/007870 and WO 2005/07624. See also, Wang and Schultz "Expanding the Genetic Code," Angewandte Chemie Int. Ed., 44 (1): 34-66 (2005). Although the following examples use an amber selector codon, they can also be used

fifteen codons of four or more bases, modifying the examples herein to include four base O-tRNA and modified synthetases to include mutations similar to those previously described for various non-natural amino acid O-RSs.

For a given system, a selector codon can also include one of the codons of three natural bases, where the one that the endogenous system does not use (or rarely uses) the codon of natural bases. For example, this includes a system that lacks an tRNA that recognizes the natural three base codon, and / or a system in which the three base codon is a rare codon.

Selector codons optionally include unnatural base pairs. These unnatural base pairs

25 further expand the existing genetic alphabet. An additional base pair increases the number of triplet codons from 64 to 125. The properties of three base pairs include stable and selective base pairing, efficient enzymatic incorporation into DNA with high fidelity by polymerase, and extension by primers continued effective after the synthesis of the unnatural base pair that is generated. Descriptions of unnatural base pairs that can be adapted for methods and compositions include, for example, Hirao, et al., (2002) "An unnatural base pair for incorporating amino acid analogues into protein," Nature Biotechnology 20: 177-182 . See also Wu et al., (2002) J. Am. Chem. Soc., 124: 14626-14630. Other related publications are indicated below.

For in vivo use, the unnatural nucleoside is membrane permeable and phosphorylates to form triphosphate.

35 correspondent. In addition, the increased genetic information is stable and cellular enzymes do not destroy it. Previous efforts made by Benner and others took advantage of hydrogen bond formation patterns that are different from those present in canonical Watson-Crick pairs, of which the most prominent example is the iso-C: iso-G pair. See, for example, Switzer et al., (1989) J. Am. Chem. Soc., 111: 8322; and Piccirilli et al., (1990) Nature 343: 33; Kool, (2000) Curr. Opin. Chem. Biol., 4: 602. These bases in general erroneously mate to a certain degree with natural bases and cannot be enzymatically replicated. Kool et al. Demonstrated that hydrophobic packing interactions between bases can replace the bonds with hydrogen bonds to direct the formation of base pairs. See Kool, (2000) Curr. Opin. Chem. Biol., 4: 602; and Guckian and Kool, (1998) Angew. Chem. Int. Ed. Engl., 36, 2825. In an effort to develop an unnatural base pair that meets all the above requirements, Schultz, Romesberg and collaborators synthesized and studied

Four. Five systematically a series of non-natural hydrophobic bases. It was observed that a PICS: PICS autopar is more stable than natural base pairs, and can be effectively incorporated into the DNA by means of the Klenow fragment (FK) of the Escherichia coli DNA polymerase I. See, for example McMinn et al., (1999) J. Am. Chem. Soc., 121: 11586; and Ogawa et al., (2000) J. Am. Chem. Soc., 122: 3274. The FK can synthesize a 3MN: 3MN autopar with sufficient efficiency and selectivity for biological function. See, for example, Ogawa et al., (2000) J. Am. Chem. Soc., 122: 8803. However, both bases act as a chain terminator for subsequent replication. Recently a mutant DNA polymerase has been developed that can be used to replicate the PICS autopar. In addition, a 7AI autopar can be replicated. See, for example, Tae et al., (2001) J. Am. Chem. Soc., 123: 7439. A new pair of metallobases has also been developed, Dipic: Py, which forms a stable pair after joining Cu (II). See Meggers et al., (2000) J. Am. Chem. Soc., 122: 10714. Since extended codons and

55 Unnatural codons are intrinsically orthogonal to natural codons, the methods of the invention can advantageously use this property to generate orthogonal tRNAs for them.

A translational derivation system can also be used to incorporate an unnatural amino acid into an unwanted polypeptide. In a translational derivation system, a large sequence is inserted into a gene but not translated into a protein. The sequence contains a structure that serves as a key to induce the ribosome jump over the sequence and resume translation downstream of the insertion.

NON-NATURAL AMINO ACIDS

65 As used herein, an unnatural amino acid refers to any amino acid, modified amino acid or amino acid analog other than selenocysteine ​​and / or pyrrolysine and the following twenty genetically encoded 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 generic structure of an alpha-amino acid is illustrated in Formula I:

An unnatural amino acid is normally any structure having Formula I, in which the R group is any substituent other than those used in the twenty natural amino acids. See, for example, Biochemistry by L. Stryer, 3rd ed. 1988, Freeman and Company, New York, for the structures of the twenty amino acids

10 natural Note that the unnatural amino acids of the invention can be naturally occurring compounds other than the twenty alpha amino acids above.

Since the unnatural amino acids of the present description normally differ from the natural amino acids in the side chain, the unnatural amino acids form amide bonds with other amino acids, for example, natural or unnatural, in the same way that they are formed in naturally occurring proteins However, unnatural amino acids have side chain groups that differ from natural amino acids.

Of particular interest herein is the unnatural amino acid sulfothyrosine (see FIG. 1). In addition to the unnatural amino acid sulfothyrosine, other unnatural amino acids may be simultaneously incorporated into a polypeptide of interest, for example, using an appropriate second O-RS / O-tRNA pair together with an orthogonal pair provided by the present invention. Many of these additional non-natural amino acids and suitable orthogonal pairs are known. See, the present description and references cited therein. For example, see Wang and Schultz "Expanding the Genetic Code," Angewandte Chemie Int. Ed., 44 (1): 34-66 (2005); Xie and Schultz, "An Expanding Genetic Code," Methods 36 (3): 227-238 (2005); Xie and Schultz, “Adding Amino Acids to the

25 Genetic Repertoire, ”Curr. Opinion in Chemical Biology 9 (6): 548-554 (2005); and Wang et al., "Expanding the Genetic Code," Annu. Rev. Biophys. Biomol Struct., 35: 225-249 (2006).

In other unnatural amino acids, for example, the R group in Formula I optionally comprises an alkyl, aryl, acyl, hydrazine, cyano, halo, hydrazide, alkenyl, ether, borate, boronate, phospho, phosphono, phosphine, enone, imine , Ester, hydroxylamine, amine and the like or any combination thereof. Other unnatural amino acids of interest include, but are not limited to, amino acids comprising a photoactivatable cross-linking agent, spin-labeled amino acids, acids, fluorescent amino acids, metal-binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids with new functional groups, amino acids. that interact covalently or non-covalently with other molecules, amino acids with photoprotective and / or photoisomerizable groups, Biotin-containing amino acids or biotin analogs, keto-containing amino acids, glycosylated amino acids, a saccharide moiety attached to the amino acid side chain, amino acids comprising polyethylene glycol or polyether, amino acids substituted with heavy atoms, chemically cleavable or photo cleavable amino acids, amino acids with an elongated side chain compared to natural amino acids (e.g., polyethers or long chain hydrocarbons, for example, of more than about 5, of

40 more than about 10 carbons, etc.), amino acids containing sugar bonded to carbon, amino acids containing aminothioacid, and amino acids containing one or more toxic residues.

In another aspect, the present description provides unnatural amino acids having the general structure illustrated by the following Formula IV:

An unnatural amino acid having this structure is normally any structure in which R1 is a substituent used in one of twenty natural amino acids (eg, tyrosine or phenylalanine) and R2 is a substituent. Therefore, this type of unnatural amino acid can be considered as a natural amino acid derivative.

In addition to unnatural amino acids that contain the sulfothyrosine structure shown in FIG. 1, an unnatural amino acid may also optionally comprise modified main chain structures, for example, as illustrated by the structures of Formula II and III:

wherein Z normally comprises OH, NH2, SH, NH-R ', or S-R'; X and Y, which may be the same or different, normally comprise S or O, and R and R ', which are optionally the same or different, are normally selected from the same list of constituents for the group R described above for unnatural amino acids that 10 have Formula I, as well as hydrogen. For example, the unnatural amino acids of the present description optionally comprise substitutions in the amino or carboxyl group, as illustrated by Formula II and

III. Unnatural amino acids of this type include, but are not limited to, α-hydroxy acids, α-thio acids, α-amino thiocarboxylates, for example, with side chains corresponding to the twenty common natural amino acids or unnatural side chains. In addition, substitutions in the α-carbon optionally include

fifteen disubstituted L, D or α-α 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 as well as 3,4,6,7,8 and 9-membered ring proline analogs, β and γ amino acids such as substituted β-alanine and γ-aminobutyric acid.

twenty In some aspects, the invention uses unnatural amino acids in the L configuration. However, it is not intended that the invention be limited to the use of unnatural amino acids in the L configuration. It is contemplated that the D-enantiomers of these unnatural amino acids.

One skilled in the art will recognize that a wide variety of analogs can easily be obtained.

25 Natural amino acids of natural origin. For example, but without limitation, unnatural derivatives of tyrosine are easily produced. Tyrosine analogs include, for example, para-substituted tyrosines, orthosubstituted tyrosines and meta-substituted tyrosines, in which the substituted tyrosine comprises an alkynyl group, an acetyl group, benzoyl group, an amino group, a hydrazine, a hydroxyamine, a thiol group, a carboxy group, an isopropyl group, a methyl group, a C6-C20 or branched straight chain hydrocarbon, a saturated hydrocarbon or

30 unsaturated, an O-methyl group, a polyether group, a nitro group, or the like. In addition, substituted multiple aryl rings are also contemplated. The glutamine analogs of the present disclosure include, without limitation, α-hydroxy derivatives, substituted γ derivatives, cyclic derivatives and amide substituted glutamine derivatives. Examples of phenylalanine analogs include, but are not limited to, para-substituted phenylalanines, ortho-substituted phenylalanines, and meta-substituted phenylalanines, wherein the substituent comprises an alkynyl group, a hydroxy group, a group

35 methoxy, a methyl group, an allyl group, an aldehyde, a nitro, a thiol group, a keto group or the like. Specific examples of natural amino acids do not include, but are not limited to, sulfothyrosine, p-ethylthiocarbonyl-L-phenylalanine, p (3-oxobutanoyl) -L-phenylalanine, 1,5-dansylalanine, amino acid 7-amino-coumarin, amino acid 7-hydroxy -coumarin, nitrobenzyl-serine, O- (2-nitrobenzyl) -L-tyrosine, p-carboxymethyl-L-phenylalanine, p-cyano-L-phenylalanine, m-cyano-Lphenylalanine, biphenylalanine, 3-amino-L-tyrosine , bipyridyl alanine, p- (2-amino-1-hydroxyethyl) -L-phenylalanine, p

40 isopropylthiocarbonyl-L-phenylalanine, 3-nitro-L-tyrosine and p-nitro-L-phenylalanine. Also a p-propargiloxyphenylalanine, a 3,4-dihydroxy-L-phenylalanine (DHP), a 3, 4, 6-trihydroxy-L-phenylalanine, a 3,4,5-tri-hydroxy-L-phenylalanine, 4- nitrophenylalanine, a p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, an L-3- (2-naphthyl) alanine, a 3-methylphenylalanine, an O-4alyl-L-tyrosine, a 4-propyl- L-tyrosine, a 3-nitro-tyrosine, a 3-thiol-tyrosine, a tri-O-acetyl-GlcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido- L-phenylalanine, a p-acyl-L-phenylalanine, a

Four. Five p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, a pbromophenylalanine, a p-amino-L-phenylalanine and an isopropyl-L-phenylalanine and the like. The structures of various unnatural amino acids are described in the references cited herein. See also, document WO 2006/110182, filed on October 27, 2005, entitled "ORTHOGONAL TRANSLATION COMPONENTS FOR THE VIVO INCORPORATION OF UNNATURAL AMINO ACIDS".

5 Chemical synthesis of unnatural amino acids

Many of the unnatural amino acids provided above are commercially available, for example from Sigma (United States) or Aldrich (Milwaukee, WI, United States). Those that are not commercially available are optionally synthesized as provided in various publications or using conventional methods known to those skilled in the art. For organic synthesis techniques, see, for example, Organic Chemistry by Fessendon and Fessendon, (1982, Second Edition, Willard Grant Press, Boston Mass.); Advanced Organic Chemistry by March (Third Edition, 1985, Wiley and Sons, New York); and Advanced Organic Chemistry by Carey and Sundberg (third edition, parts A and B, 1990, Plenum Press, New York). Additional publications describing the synthesis of unnatural amino acids include, for example, document WO 2002/085923 entitled "In vivo incorporation of Unnatural Amino Acids;" Matsoukas et al., (1995) J. Med. Chem., 38, 4660-4669; King and Kidd (1949) "A New Synthesis of Glutamine and of γ-Dipeptides of Glutamic Acid from Phthylated Intermediates," J. Chem. Soc., 3315-3319; Friedman and Chatterrji (1959) Synthesis of Derivatives of Glutamine as Model Substrates for Anti-Tumor Agents. J. Am. Chem. Soc. 81, 3750-3752; Craig et al., (1988) Absolute Configuration of the Enantiomers of 7-Chloro-4 [[4- (diethylamino) -1-methylbutyl] amino] quinoline (Chloroquine). J. Org. Chem. 53, 1167-1170; Azoulay, M., Vilmont, M. and Frappier, F. (1991) Glutamine analogues as Potential Antimalarials ,. Eur. J. Med. Chem. 26, 201-5; Koskinen, AMP & Rapoport, H. (1989) Synthesis of 4-Substituted Prolines as Conformationally Constrained Amino Acid Analogues. J. Org. Chem. 54, 1859-1866; Christie,

BD and Rapoport, H. (1985) Synthesis of Optically Pure Pipecolates from L-Asparagine. Application to the Total Synthesis of (+) - Apovincamine through Amino Acid Decarbonylation and Iminium Ion Cyclization. J. Org. Chem

25 1989: 1859-1866; Barton et al., (1987) Synthesis of Novel a-Amino-Acids and Derivatives Using Radical Chemistry: Synthesis of L- and Da-Amino-Adipic Acids, La-aminopimelic Acid and Appropriate Unsaturated Derivatives. Tetrahedron Lett. 43: 4297-4308; and, Subasinghe et al., (1992) Quisqualic acid analogues: synthesis of betaheterocyclic 2-aminopropanoic acid derivatives and their activity at a novel quisqualate-sensitized site. J. Med. Chem.

35: 4602-7. See also, International Publication WO 2004/058946, entitled "PROTEIN ARRAYS", presented on December 22, 2003.

Cellular uptake of unnatural amino acids

The uptake of unnatural amino acids by a cell is a matter that is normally considered when

35 they design and select unnatural amino acids, for example, for incorporation into a protein. For example, the high loading density of α amino acids suggests that these compounds are probably not permeable to the cell. The cell captures natural amino acids through a collection of protein-based transport systems that often have varying degrees of specificity of amino acids. A quick scan can be performed to evaluate which unnatural amino acids, if any, are captured by the cells. See, for example, toxicity tests, for example, in International Publication WO 2004/058946, entitled "PROTEIN ARRAYS," filed December 22, 2003; and Liu and Schultz (1999) Progress towards the evolution of an organism with an expanded genetic code. PNAS 96: 4780-4785. Although uptake is easily analyzed with various assays, an alternative to design unnatural amino acids that are susceptible to cell uptake pathways is to provide biosynthetic pathways to create amino acids in vivo.

Four. Five Biosynthesis of unnatural amino acids

There are already many biosynthetic pathways in cells for the production of amino acids and other compounds. Although a biosynthetic method for a particular non-natural amino acid may not exist in nature, for example, in a cell, the present description provides such methods. For example, biosynthetic pathways for unnatural amino acids can optionally be generated in host cells by adding new enzymes or by modifying existing routes of the host cell. The new additional enzymes are optionally naturally occurring enzymes or artificially developed enzymes. For example, the biosynthesis of p-aminophenylalanine (as presented in an example of WO 2002/085923) is based on the addition of a combination of enzymes

55 known from other organisms. The genes for these enzymes can be introduced into a cell by transforming the cell with a plasmid that comprises the genes. When genes are expressed in the cell, they provide an enzymatic route to synthesize the desired compound. Examples of the types of enzymes that are optionally added are provided in the examples presented below. Additional enzyme sequences are found, for example, in the Genbank. Also artificially developed enzymes are optionally added to a cell in the same way. In this way, the cellular machinery and resources of a cell are manipulated to produce unnatural amino acids.

In fact, any of several methods can be used to produce new enzymes for use in biosynthetic routes, or for the development of existing routes, for the production of non-natural amino acids in vitro or in vivo. To produce unnatural amino acids (or, in fact, to develop synthetases that have new substrate specificities or other activities of interest) many available methods can be applied to develop enzymes and other biosynthetic pathway components. For example, DNA transposition is optionally used to develop new enzymes and / or routes of said enzymes for the production of unnatural amino acids (or for the production of new synthetases), in vitro or in vivo. See, for example, Stemmer (1994), Rapid evolution of a protein in vitro by DNA shuffling, Nature 370 (4): 389-391; and, Stemmer, (1994), DNA shuffling by 5 random fragmentation and reassembly: In vitro recombination for molecular evolution, Proc. Natl. Acad. Sci. USES.,

91: 10747-10751. A related strategy combines families of related genes (for example, homologs) to rapidly develop enzymes with desired characteristics. An example of such "family gene combination" methods is found in Crameri et al. (1998) “DNA shuffling of a family of genes from diverse species accelerates directed evolution” Nature, 391 (6664): 288-291. New enzymes (components of biosynthetic pathways or synthetases) can also be generated using a DNA recombination procedure known as "incremental truncation for the creation of hybrid enzymes" ("ITCHY"), for example, as described in Ostermeier et al. (1999) "A combinatorial approach to hybrid enzymes independent of DNA homology" Nature Biotech 17: 1205. This strategy can also be used to generate a library of enzymes or other path variants that can serve as substrates for one or more in vitro or in vivo recombination methods: See also, Ostermeier et al. (1999) “Combinatorial Protein Engineering by Incremental Truncation,” Proc. Natl. Acad. Sci. United States, 96: 3562-67, and Ostermeier et al. (1999), "Incremental Truncation as a Strategy in the Engineering of Novel Biocatalysts," Biological and Medicinal Chemistry, 7: 2139-44. Another strategy uses exponential set mutagenesis to produce enzyme libraries or other route variants that are selected, for example, by the ability to catalyze a biosynthetic reaction relevant to the production of a non-natural amino acid (or a new synthetase). In this strategy, small groups of residues are randomly distributed in a sequence of interest in parallel to identify, in each altered position, amino acids that lead to functional proteins. Examples of such procedures, which can be adapted to the present invention to produce new enzymes for the production of unnatural amino acids (or new synthetases), can be found in Delegrave and Youvan (1993) Biotechnology Research 11: 1548-1552. In yet another strategy, random or semi-random mutagenesis can be used using doped or generated oligonucleotides for genetically engineered production of enzymes and / or path components, for example, using the general mutagenesis methods of, for example, Arkin and Youvan (1992) "Optimizing nucleotide mixtures to encode specific subsets of amino acids for semi-random mutagenesis" Biotechnology 10: 297-300; or Reidhaar-Olson et al. (1991) "Random mutagenesis of protein sequences using oligonucleotide cassettes" Methods Enzymol. 208: 564-86. Another strategy,

30 often referred to as "non-stochastic" mutagenesis, which uses polynucleotide reassembly and site saturation mutagenesis, can be used to produce enzymes and / or path components that can then be explored with respect to an ability to perform one or more functions of synthetase or biosynthetic pathway (for example for the production of unnatural amino acids in vivo). See, for example, Short “NON-STOCHASTIC GENERATION OF GENETIC VACCINES AND ENZYMES” document WO 00/46344.

35 An alternative to such mutational methods involves recombining entire genomes of organisms and selecting the resulting progeny for particular path functions (which is often referred to as "complete genome combination"). This strategy can be applied to the present invention, for example, by genomic recombination and selection of an organism (for example, an E. coli cell or another cell) with respect to the ability to produce

40 an unnatural amino acid (or intermediate thereof). For example, the methods described in the following publications can be applied for the design of routes for the development of existing and / or new routes in cells to produce unnatural amino acids in vivo: Patnaik et al. (2002) "Genome shuffling of lactobacillus for improved acid tolerance" Nature Biotechnology, 20 (7): 707-712; and Zhang et al. (2002) “Genome shuffling leads to rapid phenotypic improvement in bacteria” Nature, February 7, 415 (6872): 644-646.

Four. Five Other techniques are also available for genetically engineered production of metabolic organisms and pathways, for example, for the production of desired compounds, and can also be applied for the production of unnatural amino acids. Examples of publications describing production strategies by genetic route engineering include: Nakamura and White (2003) “Metabolic engineering for the microbial production of 1.3

fifty propanediol ”Curr. Opin. Biotechnol 14 (5): 454-9; Berry et al. (2002) “Application of Metabolic Engineering to improve both the production and use of Biotech Indigo” J. Industrial Microbiology and Biotechnology 28: 127-133; Banta et al. (2002) “Optimizing an artificial metabolic pathway: Engineering the cofactor specificity of Corynebacterium 2,5-diketo-D-gluconic acid reductase for use in vitamin C biosynthesis” Biochemistry, 41 (20), 6226-36; Selivonova et al. (2001) “Rapid Evolution of Novel Traits in Microorganisms” Applied and Environmental Microbiology,

55 67: 3645, and many others.

Regardless of the method used, normally the unnatural amino acid produced with a biosynthetic pathway created by genetic engineering of the invention, is produced at a concentration sufficient for effective protein biosynthesis, for example, a natural cellular amount, but not to such a degree as to affect so

60 significant to the concentration of other cellular amino acids or to deplete cellular resources. Typical concentrations produced by this method in vivo are from about 10 mM to about 0.05 mM. Once a cell is genetically engineered to produce desired enzymes for a specific route and an unnatural amino acid is generated, in vivo selections are optionally used to further optimize the production of unnatural amino acid for both the synthesis of ribosomal proteins and for he

65 cell growth

Orthogonal components to incorporate unnatural amino acids

The invention provides compositions and methods as defined in the claims for producing orthogonal components for incorporating the unnatural amino acid sulfothyrosine (see FIG. 1) into a chain.

5 growing polypeptide in response to a selector codon, for example, an amber termination codon, an antisense codon, a codon of four or more bases, etc., for example, in vivo. For example, the invention provides orthogonal tRNA (O-tRNA), orthogonal aminoacyl-tRNA synthetases (O-RS) and pairs thereof. These pairs can be used to incorporate an unnatural amino acid into growing polypeptide chains.

A composition of the invention includes an orthogonal aminoacyl tRNA aminoacyl (O-RS), wherein the O-RS preferably aminoacylates an O-tRNA with a sulfothyrosine. In certain embodiments, the O-RS comprises an amino acid sequence comprising SEQ ID Nos: 4, 6, 8 or 10, and conservative variations thereof. In certain embodiments of the invention, the O-RS preferably aminoacylates the O-tRNA over any endogenous tRNA with a particular unnatural amino acid, in which the O-RS has a predilection for O

fifteen TRNA, and in which the ratio of the O-tRNA loaded with an unnatural amino acid with respect to the endogenous tRNA loaded with the same unnatural amino acid is greater than 1: 1, and more preferably in which the O-RS loads the O -RNA exclusively or almost exclusively.

A composition that includes an O-RS may optionally additionally include an orthogonal tRNA (O-tRNA), in which the O-tRNA recognizes a selector codon. Typically, an O-tRNA of the invention may include at least about 45%, 50%, 60%, 75%, 80% or 90% or more of suppression efficiency in the presence of a related synthetase in response to a selector codon compared to the O-tRNA that comprises or is encoded by a polynucleotide sequence as indicated in the sequence list and the examples herein. In one embodiment, the suppression efficiency of the O-RS and the O-tRNA

25 together it is, for example, 5 times, 10 times, 15 times, 20 times or 25 times or more times greater than the suppression efficiency of the O-tRNA lacking the O-RS. In some aspects, the suppression efficacy of the O-RS and the O-tRNA together is at least 45% of the suppression efficacy of an orthogonal pair of tyrosyl tRNA synthetase derived from Methanococcus jannaschii.

A composition that includes an O-tRNA may optionally include a cell (for example, a eubacteria cell, such as an E. coli cell and the like, or a eukaryotic cell such as a yeast cell) and / or a translation system .

The invention also provides a cell (for example a eubacterium cell or a yeast cell) that

35 it comprises a translation system, as defined in the claims, wherein the translation system includes an orthogonal tRNA (O-tRNA); an orthogonal aminoacyl-tRNA synthetase (O-RS); and an unnatural amino acid sulfothyrosine. Normally, the O-RS preferably aminoaclates the O-tRNA over any endogenous tRNA with the unnatural amino acid, in which the O-RS has a predilection for the O-tRNA, and in which the proportion of the O-tRNA loaded with the unnatural amino acid with respect to the endogenous tRNA loaded with the unnatural amino acid is greater than 1: 1, and more preferably in which the O-RS charges the O-tRNA exclusively or almost exclusively. The O-tRNA recognizes the first selector codon, and the O-RS preferably aminoaclates the O-tRNA with an unnatural amino acid. In one embodiment, the O-tRNA comprises or is encoded by a polynucleotide sequence as indicated in SEQ ID NO: 1, or a complementary polynucleotide sequence thereof. In one embodiment, the O-RS comprises an amino acid sequence as indicated in SEQ ID Nos: 4, 6, 8 or 10, and variations

Four. Five Conservative of it.

A cell of the invention may also optionally comprise an additional different O-tRNA / O-RS pair and a second unnatural amino acid, for example, where this O-tRNA recognizes a second selector codon and this O-RS preferably aminoaclates the O -RNA corresponding to the second non-natural amino acid, where the second amino acid is different from the first non-natural amino acid. Optionally, a cell of the invention includes a nucleic acid comprising a polynucleotide encoding a polypeptide of interest, wherein the polynucleotide comprises a selector codon that is recognized by the O-tRNA.

In certain embodiments, a cell of the invention is a eubacterial cell (such as E. coli), which includes

55 an orthogonal tRNA (O-tRNA), an orthogonal aminoacyl-tRNA synthetase (O-RS), an unnatural amino acid, and a nucleic acid comprising a polynucleotide encoding a polypeptide of interest, where the polynucleotide comprises the selector codon that is recognized by the O-tRNA. In certain embodiments of the invention, the O-RS preferably aminoacylates the O-tRNA with the unnatural amino acid with an efficacy that is greater than the efficiency with which the O-RS aminoacylates any endogenous tRNA.

In certain embodiments of the invention, an O-tRNA of the invention comprises or is encoded by a polynucleotide sequence as indicated in the sequence lists (for example SEQ ID NO: 1) or in the examples herein, or a complementary polynucleotide sequence thereof. In certain embodiments of the invention, an O-RS comprises an amino acid sequence as indicated in the sequence list, or a conservative variation thereof. In one embodiment, the O-RS, or a part thereof, is encoded by a polynucleotide sequence encoding an amino acid as indicated in the list.

of sequences or in the examples herein, or a complementary polynucleotide sequence thereof.

The O-tRNA and / or the O-RS of the invention can be derived from any of several organisms (for example, 5 eukaryotic and / or non-eukaryotic organisms).

As defined in the claims, polynucleotides are also a feature of the invention. A polynucleotide of the invention (for example SEQ ID Nos: 5, 7, 9 or 11) includes an artificial polynucleotide (for example, man-made and not of natural origin) comprising a nucleotide sequence encoding a polypeptide as indicated in the sequence list herein and / or is complementary to that polynucleotide sequence. A polynucleotide of the invention may also include a nucleic acid that hybridizes with a polynucleotide described above, under conditions of high stringency, along substantially the entire length of the nucleic acid. A polynucleotide of the invention also includes a polynucleotide having an identity of, for example, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or more

fifteen with that of a naturally occurring tRNA or a corresponding coding nucleic acid (but a polynucleotide of the invention is different from a naturally occurring tRNA or corresponding coding nucleic acid) where the tRNA recognizes a selector codon, for example, a codon of four bases. Also included in the polynucleotides of the invention are artificial polynucleotides having an identity of at least 80%, at least 90%, at least 95%, at least 98% or more with any of the foregoing and / or a polynucleotide comprising a conservative variation of any of the above.

Also characteristic of the invention are vectors that comprise a polynucleotide of the invention, as defined in the claims. For example, a vector of the invention may include a plasmid, a cosmid, a phage, a virus, an expression vector and / or the like. Also a feature of the invention is a cell that

25 It comprises a vector of the invention.

Methods of producing components of an O-tRNA / O-RS pair are also features of the invention. The components produced by these methods are also a feature of the invention. For example, methods to produce at least one tRNA that is orthogonal to a cell (O-tRNA) include generating a library of mutant tRNAs, mutating an anticodon loop of each member of the mutant tRNA library to allow recognition of a codon. selector, thereby providing a library of potential O-tRNAs, and negatively selecting a first population of cells of a first species, where the cells comprise a member of the potential tRNA library. Negative selection removes cells that comprise a member of the potential O-tRNA library that is aminoacylated by an aminoacyl-tRNA

35 synthetase (RS) that is endogenous to the cell. This provides a set of tRNAs that are orthogonal to the cell of the first species, thereby providing at least one O-tRNA. An O-tRNA produced by the methods of the invention is also provided.

In certain embodiments the methods further comprise subjecting a second population of cells of the first species to positive selection, where the cells comprise a member of the set of tRNAs that are orthogonal to the cell of the first species, a related aminoacyl-tRNA synthetase, and a positive selection marker. Using the positive selection, cells comprising a member of the tRNA set that is aminoacylated by the related aminoacyl-tRNA synthetase and that shows a desired response in the presence of the positive selection marker are provided, thereby providing an O- TRNA In certain

Four. Five embodiments, the second population of cells comprises cells that have not been removed by negative selection.

Methods for identifying an orthogonal aminoacyl tRNA synthetase loading a natural amino acid are also provided. For example, the methods include selecting a population of cells of a first species, where each cell comprises: 1) a member of a plurality of aminoacyl-tRNA synthetases (RS), (for example, the plurality of RS may include mutant RS, RS derived from a different species of a first species or both mutant RS or as RS from a different species of a first species); 2) orthogonal tRNA (O-tRNA) (for example, of one or more species); and 3) a polynucleotide encoding a positive selection marker and comprising at least one selector codon.

55 Cells (for example a host cell) that show potentiation in suppression efficiency are selected or identified compared to cells lacking or having a reduced amount of the member of the plurality of RS. These selected / identified cells comprise an active RS that aminoacylates the O-tRNA. Also a feature of the invention is an orthogonal aminoacyl-tRNA synthetase identified by the method of the invention.

Also characteristic of the invention are methods for producing a protein in a cell (for example, in a eurobacterial cell, such as an E. coli cell, or the like, or in a yeast cell) having the unnatural amino acid in a selected position. For example, one method includes allowing a cell to grow, in an appropriate medium, where the cell comprises a nucleic acid comprising at least one selector codon and encoding a protein, providing the unnatural amino acid, and incorporating the unnatural amino acid into the position

specified in the protein during translation of the nucleic acid with said at least one selector codon, thereby producing the protein. The cell further comprises: an orthogonal tRNA (O-tRNA) that functions in the cell and recognizes the selector codon; and, an orthogonal aminoacyl-tRNA synthetase (O-RS) that preferably aminoacylates the O-tRNA with the unnatural amino acid. A protein produced by this method

5 It is also a feature of the invention. Of particular interest are methods for producing the sulfated form of hirudin, which is used as an anticoagulant.

The invention also provides compositions that include proteins, wherein the proteins comprise sulfothyrosine. In certain embodiments, the protein may comprise an amino acid sequence that has

10 an identity of at least 75% with that of a known protein, for example, hirudin, a therapeutic protein, a diagnostic protein, an industrial enzyme or a part thereof. Optionally, the composition comprises a pharmaceutically acceptable carrier.

SEQUENCES OF NUCLEIC ACIDS AND POLYPEPTIDES AND VARIANTS

fifteen As described herein, the invention provides polynucleotide sequences as defined in the claims encoding, for example, tRNAs and O-RSs and amino acid sequences of polypeptides, for example, O-RS and, for example. , compositions, systems and methods comprising said polynucleotide or polypeptide sequences. This document describes examples of such

twenty sequences, for example, O-tRNA and O-RS and amino acid and nucleotide sequences (see FIG. 7, for example, SEQ ID Nos: 1 and 4-11). However, one skilled in the art will appreciate that the invention is not limited to these sequences described herein, for example, in the Examples and in the sequence list. An expert will appreciate that the invention also provides many sequences related to the functions described herein, for example, polynucleotides and polypeptides encoding conservative variants of an O-RS

25 described in this document.

The construction and analysis of orthogonal synthetase (O-RS) species that are capable of aminoacylating an O-tRNA with a sulfothyrosine are described in Example 1. This Example describes the construction and analysis of O-RS species that are capable of incorporating the unnatural amino acid sulfothyrosine.

30 The invention provides polypeptides (O-RS) and polynucleotides, for example, O-tRNA, polynucleotides encoding O-RS or parts thereof, oligonucleotides used to isolate aminoacyl-tRNA synthetase clones, etc. The polynucleotides of the invention include those that encode proteins or polypeptides of interest of the invention with one or more selector codons. In addition, the polynucleotides of the invention include, for example, a

35 polynucleotide comprising a nucleotide sequence as indicated in SEQ ID Nos: 5, 7, 9 or 11, and a polynucleotide that is complementary to, or encoding, a polynucleotide sequence thereof. A polynucleotide of the invention also includes any polynucleotide encoding an O-RS amino acid sequence comprising SEQ ID Nos: 4, 6, 8 or 10. Similarly, an artificial nucleic acid that hybridizes with a polynucleotide indicated above under conditions of high stringency along substantially all of the

40 Length of a nucleic acid (and is different from a naturally occurring polynucleotide) is a polynucleotide of the invention. In one embodiment, a composition includes a polypeptide of the invention and an excipient (eg, buffer, water, pharmaceutically acceptable excipient, etc.). The invention also provides an antibody or antisera specifically immunoreactive with a polypeptide of the invention. An artificial polynucleotide is a polynucleotide that is manufactured by man and is not naturally occurring.

Four. Five A polynucleotide of the invention also includes an artificial polynucleotide having an identity of, for example, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or greater with a naturally occurring tRNA (but is different from a naturally occurring tRNA) or any tRNA or nucleic acid encoding it in a list or example herein. A polynucleotide also includes a

fifty artificial polynucleotide that has an identity of, for example, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or greater (but not 100% identity) with that of an tRNA of natural origin.

In certain embodiments, a vector (for example, a plasmid, a cosmid, a phage, a virus, etc.) comprises a polynucleotide of the invention. In one embodiment, the expression vector may be an expression vector. In

55 Another embodiment of the expression vector may include a promoter operably linked to one or more polynucleotides of the invention. In another embodiment, a cell comprises a vector that includes a polynucleotide of the invention.

One skilled in the art will also appreciate that many variants of the described sequences are included in the invention. For example, conservative variations of the sequences described are included in the invention.

60 They provide a functionally similar sequence. Variants of the nucleic acid polynucleotide sequences are considered to be included in the invention, in which the variants hybridize with at least one described sequence and recognize a selector codon. Also included in the invention are unique sub-sequences of the sequences described herein as determined, for example, by conventional sequence comparison techniques.

65 Conservative Variations

Due to the degeneracy of the genetic code, "silent substitutions" (ie, substitutions in a nucleic acid sequence that do not result in an alteration in an encoded polypeptide) are a

5 implicit characteristic of all nucleic acid sequences encoding an amino acid. Similarly, in the "conservative amino acid substitutions", one or a few amino acids of an amino acid sequence have been replaced by different amino acids with very similar properties, they are also simply identified as very similar to a described construction. Said conservative variations of each described sequence are a feature of the present invention.

10 "Conservative variations" of a particular nucleic acid sequence refer to those nucleic acids encoding identical or essentially identical amino acid sequences or, when the nucleic acid does not encode an amino acid sequence, to essentially different sequences. An expert will recognize that individual substitutions, deletions or additions that alter, add or eliminate a single amino acid or a small

fifteen Percentage of amino acids (usually less than 5%, more normally less than 4%, 2% or 1%) in an encoded sequence are "conservatively modified variations", where alterations cause the deletion of an amino acid, the addition of a amino acid or the substitution of an amino acid with a chemically similar amino acid. Thus, "conservative variations" of a polypeptide sequence listed in the present invention include substitutions of a small percentage, usually less than 5%, plus

twenty normally less than 2% or 1%, of the amino acids of the polypeptide sequence, with an amino acid of the same conservative substitution group. Finally, the addition of sequences that do not alter the encoded activity of the nucleic acid molecule, such as the addition of a non-functional sequence, is a conservative variation of the basic nucleic acid.

25 Conservative substitution tables that provide functionally similar amino acids are well known in the art, where one amino acid residue is replaced by another amino acid residue that has similar chemical properties (eg, aromatic side chains or positively charged side chains) and, therefore, therefore, it does not substantially change the functional properties of the polypeptide molecule. Below are illustrative groups containing natural amino acids with similar chemical properties, where substitution within

30 A group is a "conservative substitution."

Conservative amino acid substitutions

<dl><dt>Non-polar and / or Aliphatic Side Chains </dt><dd>Polar Side Chains, Not Loaded Aromatic Side Chains Positively Loaded Side Chains Side Loads Negatively Loaded </dd></dl>

<dl><dt>Glycine Alanine Valine Leucine Isoleucine Proline </dt><dd>Serine Threonine Cysteine ​​Methionine Asparagine Glutamine Phenylalanine Tyrosine Tryptophan Lysine Arginine Histidine Aspartate Glutamate </dd></dl>

35 Nucleic acid hybridization

To identify nucleic acids of the invention comparative hybridization can be used, including conservative variations of nucleic acids of the invention, and this comparative hybridization method is a preferred method for differentiating nucleic acids of the invention. In addition, nucleic acids are a feature of the invention.

40 target that hybridize with a nucleic acid represented by SEQ ID Nos: 5, 7, 9 or 11, under conditions of high, ultra-high and ultra-high stringency. Examples of such nucleic acids include those with one or a few conservative or silent nucleic acid substitutions compared to a given nucleic acid sequence.

Four. Five It is said that a test nucleic acid specifically hybridizes with a probe nucleic acid when it hybridizes at least 50% well with the probe as with the perfectly matched complementary target, that is, with a signal-to-interference ratio of at least half as high as the hybridization of the probe with the target in conditions where the perfectly matched probe binds to the complementary target perfectly coincident with a relationship between signal interference that is at least from about 5 times to 10 times as

fifty high as that observed for hybridization with any of the unpaired target nucleic acids.

Nucleic acids "hybridize" when they are associated, usually in solution. Nucleic acids hybridize due to various well-characterized physicochemical forces, such as hydrogen bond formation, solvent exclusion, base stacking and the like. A comprehensive guide to nucleic acid hybridization is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes, Part I, Chapter 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assays, ”(Elsevier, New York), as well as in Current Protocols in Molecular Biology, Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley and Sons, Inc., (attached throughout the

5 2006); Hames and Higgins (1995) Gene Probes 1 IRL Press at Oxford University Press, Oxford, England; and Hames and Higgins (1995) Gene Probes 2 IRL Press at Oxford University Press, Oxford, England, provide details on the synthesis, labeling, detection and quantification of DNA and RNA, including oligonucleotides.

An example of stringent hybridization conditions for the hybridization of complementary nucleic acids that

10 they have more than 100 complementary residues in a filter in a Southern or Northern transfer is 50% formalin with 1 mg of heparin at 42 ° C, hybridization being performed overnight. An example of rigorous washing conditions is a wash with 0.2x SSC at 65 ° C for 15 minutes (for a description of SSC buffer see, Sambrook et al., Molecular Cloning - A Laboratory Manual (3rd Ed.), Vol. 1 -3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2001). Often, high rigor washing is preceded by a washing of

fifteen Low rigor to eliminate the background signal of the probe. An example of the low stringency wash is 2x SSC at 40 ° C for 15 minutes. In general, a ratio between signal and interference 5 times greater (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.

twenty "Rigorous hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent and are different with different environmental parameters. An extensive guide to nucleic acid hybridization is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes, Part I, Chapter 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assays, ”

25 (Elsevier, New York); Hames and Higgins (1995) Gene Probes 1 IRL Press at Oxford University Press, Oxford, England; and Hames and Higgins (1995) Gene Probes 2 IRL Press at Oxford University Press, Oxford, England. Rigorous hybridization and washing conditions can be determined empirically simply for any test nucleic acid. For example, to determine stringent hybridization and washing conditions, hybridization and washing conditions are gradually increased (for example, by increasing the temperature,

30 decreasing the salt concentration, increasing the concentration of detergent and / or increasing the concentration of organic solvents such as formalin in hybridization or washing), until a selected set of criteria is met. For example, under conditions of high stringency hybridization and washing, hybridization and washing conditions are gradually increased until a probe binds to a complementary target perfectly matched with a signal-to-interference ratio that is at least 5 times that observed for

35 probe hybridization with an unpaired target.

"Very stringent" conditions are selected to be equal to the thermal melting point (Tm) of a particular probe. The Tm is the temperature (with defined ionic strength and pH) at which 50% of the test sequence hybridizes with a perfectly matched probe. For the purposes of the present invention, it is generally

40 select the "highly stringent" hybridization and washing conditions to be approximately 5 ° C less than the Tm for the specific sequence at a defined ionic strength and pH.

Hybridization and washing conditions of "ultra high stringency" are those in which the stringency of the hybridization and washing conditions are increased until the ratio between signal and interference to bind the probe

Four. Five the perfectly matched complementary target nucleic acid is at least 10 times greater than that observed for hybridization with any of the unpaired target nucleic acids. It is said that a target nucleic acid that hybridizes with a probe under such conditions, with a signal to interference ratio of at least half of that of the perfectly matched complementary target nucleic acid, binds to the probe under ultra-high stringency conditions. .

fifty Similarly, even higher stringency levels can be determined by gradually increasing the hybridization and / or washing conditions of the relevant hybridization assay. For example, those in which the stringency of the hybridization and washing conditions are increased until the ratio between signal and interference for the binding of the probe to the perfectly matched complementary target nucleic acid is at least 10

55 times, 20 times, 50 times, 100 times, 500 times or more times that observed for hybridization with any of the unpaired target nucleic acids. It is said that a target nucleic acid that hybridizes with a probe under such conditions, with a signal-to-interference ratio of at least half of that of the perfectly matched complementary target nucleic acid, binds to the probe under ultra-strict stringency conditions. high.

60 Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This happens, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy allowed by the genetic code.

Unique sub-sequences

In some aspects, the present description provides a nucleic acid comprising a unique sub-sequence in a nucleic acid selected from the O-tRNA and O-RS sequences described herein. The

5 Unique sub-sequence is unique compared to a nucleic acid corresponding to any previously known O-tRNA or O-RS nucleic acid sequence. Alignment can be performed using, for example, a BLAST setting for default parameters. Any single subsequence is useful, for example, as a probe to identify the nucleic acids of the invention.

10 Similarly, the present invention includes a polypeptide comprising a unique sub sequence in a polypeptide selected from the sequences of the O-RSs described herein. In this case, the unique subsequence is unique compared to a polypeptide corresponding to any previously known RS sequence.

fifteen The invention also provides target nucleic acids that hybridize under stringent conditions with a single coding oligonucleotide encoding a unique sub-sequence in a polypeptide selected from the O-RS sequences where the single sub-sequence is unique compared to a polypeptide corresponding to any of the polypeptides. of control (for example, parental sequences from which the synthetases of the invention were obtained, for example by mutation). Unique sequences are determined as noted.

twenty previously.

Comparison, identity and sequence homology

The expressions "identical" or "percent identity", in the context of two or more nucleic acids or sequences

25 polypeptide, refers to two or more sequences or sub-sequences that are equal or that have a specified percentage of amino acid or nucleotide residues that are equal when compared and aligned for maximum correspondence, as measured using one of two comparison algorithms. sequence described below (or other algorithms available to those skilled in the art) or by visual inspection.

30 The term "substantially identical", in the context of two nucleic acids or polypeptides (eg, DNA encoding an O-tRNA or an O-RS, or the amino acid sequence of an O-RS), refers to two or more sequences or sub-sequences having an identity of amino acid or nucleotide residues of at least about 60%, about 80%, about 90-95%, about 98%, about 99% or more, when compared and aligned for maximum correspondence, as measured

35 using a sequence comparison algorithm or by visual inspection. Such "substantially identical" sequences are normally considered "homologous", without reference to actual ancestry. Preferably, the "substantial identity" exists along a region of the sequences that has a length of at least about 50 residues, more preferably along a region of at least about 100 residues, and much more preferably, the sequences are substantially identical over at least

40 approximately 150 residues, or along the full length of the two sequences to be compared.

Proteins and / or protein sequences are "homologous" when derived, naturally or artificially, from a common ancestral protein or protein sequence. Similarly, nucleic acids and / or nucleic acid sequences are homologous when they derive, naturally or artificially, from a nucleic acid or a common ancestral nucleic acid sequence. For example, any naturally occurring nucleic acid can be modified by any available mutagenesis method to include one or more selector codons. When expressed, this mutated nucleic acid encodes a polypeptide comprising one or more unnatural amino acids. The mutation process can, of course, further alter one or more conventional codons, thus also changing one or more conventional amino acids in the resulting mutant protein. The homology is generally deduced from the sequence similarity between two or more nucleic acids or proteins (or sequences thereof). The exact percentage of sequence similarity that is useful for establishing homology varies with the nucleic acid and the protein in question, but routinely, only 25% sequence similarity is used to establish homology. Higher levels of sequence similarity can also be used, for example, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more,

55 to establish homology. Methods for determining similarity percentages (for example BLASTP and BLASTN using default parameters) are described herein and are generally available.

For sequence comparison and homology determination, normally a sequence acts as a reference sequence with which the test sequences are compared. When using an algorithm of

60 sequence comparison, the test and reference sequences are introduced into a computer, the sub-sequence coordinates are designed, if necessary, and sequence algorithm program parameters are designed. Next, the sequence comparison algorithm calculates the percentage of sequence identity for the test sequence (or sequences) with respect to the reference sequences based on the designed program parameters.

65 Optimal sequence alignment can be performed for comparison, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math 2: 482 (1981), using the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48: 443 (1970), by the similarity search method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 (1988), by means of computer applications of these

5 algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally Current Protocols in Molecular Biology, Ausubel et al. , eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley and Sons, Inc., affiliated throughout 2006).

An example of an algorithm that is suitable for determining the percentage of sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol., 215: 403-410 (1990). The BLAST analysis software is publicly available from the National Center for Biotechnology Information. This algorithm involves first identifying pairs of high-scoring sequences (HSP, High Scoring Sequence Pairs) by identifying short words of length W in the problem sequence, which fit or meet some 15 positively valued threshold T-score when aligned with a word of the same length in a sequence of databases. The neighboring word punctuation threshold is called T (Altschul et al., J. Mol. Biol., 215: 403-410 (1990). These initial neighbor word successes act as seeds to initiate searches to find the longest HSPs that contain them. Next, word hits extend in both directions along each sequence until the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always&gt; 0) and N (penalty score for mismatched remains; always &lt;0). For amino acid sequences a scoring matrix is ​​used to calculate the cumulative score. The extension of word successes in each direction stops when: the cumulative alignment score falls by the amount X from its maximum value achieved; the score

25 Cumulative tends to zero or a lower value, due to the accumulation of one or more alignments of negative scoring residues; or the end of any sequence is reached. The parameters W, T and X of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses by default a word length (W) of 11, a hope (E) of 10, a limit of 100, M = 5, N = -4 and a comparison of the two strings . For amino acid sequences, the BLASTP program uses by default a word length (W) of 3, a hope (E) of 10 and the BLOSUM62 score matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915).

In addition to calculating the percentage of sequence identity, the BLAST algorithm performs a statistical analysis of the similarity between two sequences, (see, for example, Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787

35 (1993)). A measure of the similarity provided by the BLAST algorithm is the smallest sum probability (P (N)), which provides an indication of the probability that a coincidence between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence without the smallest sum probability in a comparison of the test nucleic acid with the reference nucleic acid is less than about 0.1, more preferably less than about 0.01 and much more preferably less than about 0.001.

Mutagenesis and other molecular biology techniques

The polynucleotides and polypeptides of the invention and used therein can be manipulated using techniques of

Four. Five molecular biology. General texts describing molecular biology techniques include Berger and Kimmel, "Guide to Molecular Cloning Techniques," Methods in Enzymology, volume 152 Academic Press, Inc., San Diego, CA; Sambrook et al., Molecular Cloning - A Laboratory Manual (3rd ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2001, and Current Protocols in Molecular Biology, Ausubel et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley and Sons, Inc., (attached throughout 2003) (Ausubel). These texts describe mutagenesis, the use of vectors, promoters and many other relevant aspects related, for example, to the generation of genes that include selector codons for the production of proteins that include unnatural amino acids, orthogonal tRNAs, orthogonal synthetases and pairs of same.

55 Various types of mutagenesis are used in the invention, for example, to mutate tRNA molecules, to produce tRNA libraries, to produce synthetase libraries, or to insert selector codons that encode an unnatural amino acid in a protein or polypeptide of interest. . These include, but are not limited to, targeted random point mutagenesis, homologous recombination, DNA transposition or other methods of recursive mutagenesis, chimeric construction, mutagenesis using uracil containing templates, oligonucleotide-directed site mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using discontinuous duplex DNA or the like or any combination thereof. Other suitable methods include timely repair of mismatch, mutagenesis using repair-deficient host strains, restriction selection and restriction purification, deletion mutagenesis, total gene synthesis mutagenesis, double chain breakage repair or the like. Also included in the present invention, for example, is the

65 mutagenesis that involves chimeric constructs. In one embodiment, mutagenesis can be directed by known information of the naturally occurring molecule or of the altered or mutated natural molecule, for example, by sequence information, sequence comparisons, physical properties, crystalline structure or Similary.

Host cells are created by genetic engineering (for example, they are transformed, transduced or transfected)

5 with the polynucleotides of the invention or with constructs that include a polynucleotide of the invention, for example, a vector of the invention, which may be, for example, a cloning vector or an expression vector. For example, the coding regions for orthogonal tRNA, orthogonal tRNA synthetase and the protein to be derivatized are operably linked to gene expression control elements that are functional in the desired host cell. Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences and promoters useful for the regulation of the expression of the particular target nucleic acid. The vectors optionally comprise generic expression cassettes that contain at least one independent terminator sequence, sequences that allow for replication 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 / or integration in

fifteen prokaryotes, eukaryotes or, preferably, in both. See Giliman and Smith, Gene 8: 81 (1979); Roberts, et al., Nature, 328: 731 (1987); Schneider et al., Protein Expr. Purif., 6435: 10 (1995); Berger and Kimmel, "Guide to Molecular Cloning Techniques," Methods in Enzymology, volume 152 Academic Press, Inc., San Diego, CA; Sambrook et al., Molecular Cloning - A Laboratory Manual (3rd ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2001, and Current Protocols in Molecular Biology, Ausubel et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley and Sons, Inc., (attached throughout 2006). The vector may be, for example, in the form of a plasmid, a bacterium, a virus, a naked polynucleotide or a conjugated polynucleotide. Vectors are introduced into cells and / or microorganisms by conventional methods including electroporation (From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985), viral vector infection, high-speed ballistic penetration with particles small with the nucleic acid inside the matrix of

25 small pearls or particles or on the surface (Klein et al., Nature 327, 70-73 (1987)), and / or the like.

For the specific site incorporation of natural amino acids in proteins without response to the amber termination codon (UAG) in E. coli, a versatile and highly effective single plasmid system was developed. In the new system, the ARNtyr (CUA) and tyrosyl-tRNA synthetase suppressor pair of M. jannaschii is encoded in a single plasmid, which is compatible with most E. coli expression vectors. A monocistronic tRNA operon was constructed under the control of the proK promoter and terminator for optimal secondary structure and tRNA processing. The introduction of a mutated form of the glnS promoter for synthetase resulted in a significant increase in suppression and fidelity efficacy. Increases in suppression efficiency were also obtained by multiple copies of tRNA gene as well as by specific mutation (D286R) in synthetase

35 (Kobayashi et al., "Structural basis for orthogonal tRNA specificities of tyrosyl-tRNA synthetases for genetic code expansion," Nat. Struct. Biol., 10 (6): 425-432 [2003]). The generality of the optimized system was also demonstrated by a highly efficient and accurate incorporation of several different non-natural amino acids, whose exclusive utilities in the study of protein function and structure were previously approved.

The ATCC provides a catalog of Bacteria and Bacteriophages useful for cloning, for example, the ATCC Catalog of Bacteria and Bacteriophages (1996) Ghema et al. (eds) published by the ATCC. Additional basic procedures for sequencing, cloning and other aspects of molecular biology and fundamental theoretical considerations are also found in Sambrook et al., Molecular Cloning - A Laboratory Manual (3rd ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2001; Current Protocols in Molecular Biology,

Four. Five Ausubel et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley and Sons, Inc., (ascribed throughout 2006), and in Watson et al. (1992) Recombinant DNA, Second Ed., Scientific American Books, NY. In addition, essentially any nucleic acid (and virtually any labeled nucleic acid, conventional or unconventional) can be ordered at the customer's request or conventionally from any of several commercial sources, such as the Midland Certified Reagent Company, The Great American Gene Company ( Ramona, CA), ExpressGen Inc. (Chicago, IL), Operon Technologies Inc. (Alameda, CA) and many others.

Host cells created by genetic engineering can be grown in conventional nutrient media modified appropriately for activities such as, for example, identification steps, activation of

55 promoters or selection of transformants. These cells can optionally be cultured in transgenic organisms. Other useful references, for example, for the isolation and culture of cells (for example, for the subsequent isolation of nucleic acids) include Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley Liss, New York and references cited in this document; Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley and 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 and Parks (eds) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL.

PROTEINS AND POLYPEPTIDES OF INTEREST

65 As defined in the claims, methods for producing a protein in a cell with an unnatural amino acid in a specific position are also a feature of the invention. For example, one method includes culturing, in an appropriate medium, the cell, in which the cell comprises a nucleic acid that comprises at least one selector codon and encodes a protein; and, provide the unnatural amino acid; in which the cell further comprises; an orthogonal tRNA (O-tRNA) that works in the cell and knows the codon

5 selector; and, an orthogonal aminoacyl-tRNA synthetase (O-RS) that preferably aminoacylates the O-tRNA with the unnatural amino acid. It is also a feature of the invention a protein produced by this method.

In certain embodiments, the O-RS comprises a predilection for aminoacylation of the related O-tRNA over any endogenous tRNA in an expression system. The relative ratio between O-tRNA and the endogenous tRNA that is loaded by the O-RS, when the O-tRNA and the O-RS are present at equimolar concentrations, may be greater than 1: 1, preferably at least about 2 : 1, more preferably 5: 1, even more preferably 10: 1, even more preferably 20: 1, even more preferably 50: 1, even more preferably 75: 1, even more preferably 95: 1, 98: 1, 99: 1, 100: 1, 500: 1, 1,000: 1, 5,000: 1 or a larger proportion.

fifteen The invention also provides compositions that include proteins, wherein the proteins comprise an unnatural amino acid. In certain embodiments, the protein may comprise an amino acid sequence that has an identity of at least 75% with that of a therapeutic protein, a diagnostic protein, an industrial enzyme or a portion thereof.

The compositions of the invention and the compositions manufactured by the methods of the invention are optionally present in a cell. The O-tRNA / O-RS pairs or the individual components of the invention can therefore be used in a host system translation machinery, which results in the incorporation of an unnatural amino acid into a protein. WO International Publication Numbers

25 2004/094593, filed on April 16, 2004, entitled "EXPANDING THE EUKARYOTIC GENETIC CODE", and WO 2002/085923, entitled "IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS", describe this process. For example, when an O-tRNA / O-RS pair is introduced into a host, for example, an Escherichia coli cell, the pair leads to the in vivo incorporation of an unnatural amino acid, such as sulfotyrosine, into a protein in response to a codon selector. The unnatural amino acid that is added to the system can be a synthetic amino acid, such as a derivative of a phenylalanine or tyrosine, which can be added exogenously to the culture medium. Optionally, the compositions of the present invention may be in an in vitro translation system or an in vivo system (or systems).

A cell of the invention provides the ability to synthesize proteins comprising non-amino acids.

35 natural in large quantities useful. In some aspects, the composition optionally includes, for example, at least 10 micrograms, at least 50 micrograms, at least 75 micrograms, at least 100 micrograms, at least 200 micrograms, at least 250 micrograms, at least 500 micrograms, at least 1 milligram, at least 10 milligrams or more of the protein comprising an unnatural amino acid or an amount that can be achieved with the in vivo protein production methods (details of the production and purification of recombinant proteins are provided herein). In another aspect, the protein is optionally present in the composition at a concentration of, for example, at least 10 micrograms of protein per liter, at least 50 micrograms of protein per liter, at least 75 micrograms of protein per liter, at least 100 micrograms of protein per liter, at least 200 micrograms of protein per liter, at least 250 micrograms of protein per liter, at least 500 micrograms of protein per liter, at least 1 milligram of protein per liter, or at least 10 milligrams

Four. Five of protein per liter or more, for example, in a cell lysate, a buffer, a pharmaceutical buffer or other liquid suspension (for example, in a volume of, for example, any from about 1 nl to about 100 l). A feature of the present description is the production of large amounts (for example, higher than normally possible with other methods, for example, in vitro translation) of a protein in a cell that includes at least one unnatural amino acid.

The incorporation of an unnatural amino acid can be done, for example, to adjust changes in the structure and / or function of the protein, for example, to change the size, acidity, nucleophilia, hydrogen bond formation, hydrophobia , accessibility of protease target sites, targeting to a moiety (for example, for a protein matrix), incorporation of markers or reactive groups, etc. Proteins that include a

55 Unnatural amino acid may have improved or even completely new catalytic or physical properties. For example, by including a non-natural amino acid in a protein, the following properties are optionally modified: toxicity, biodistribution, structural properties, spectroscopic properties, chemical and / or photochemical properties, catalytic capacity, half-life (for example, serum half-life) , ability to react with other molecules, for example, in a covalent or non-covalent manner and the like. Compositions that include proteins that include at least one unnatural amino acid are useful for, for example, new therapeutic, diagnostic, catalytic, industrial enzymes, binding proteins (eg, antibodies), and for example, for the study of structure and function of proteins. See, for example, Dougherty, (2000) Unnatural Amino Acids as Probes of Protein Structure and Function, Current Opinion in Chemical Biology, 4: 645-652.

65 In some aspects of the present description, a composition includes at least one protein with at least one, for example, at least two, at least three, at least four, at least five, at least six, at least seven, at least

eight, at least nine or at least ten or more unnatural amino acids. Unnatural amino acids may be the same or different, for example, there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different sites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different unnatural amino acids. In another aspect, a composition includes a protein in which at least one, but not all, the particular amino acids present in the protein is an unnatural amino acid. For a given protein with more than one unnatural amino acid, the unnatural amino acids may be identical or different (for example, the protein may include two or more different types of unnatural amino acids, or it may include two of the same unnatural amino acids) . For a given protein with more than two unnatural amino acids, the unnatural amino acids may be the same, different or a combination of a multiple non-natural amino acid of the same type with at least one non-amino acid.

10 natural different.

Using the compositions and methods herein, essentially any protein (or part thereof) that includes an unnatural amino acid (and any corresponding coding nucleic acid, including, for example, one or more selector codons) can be produced. No attempt has been made to identify the hundreds

fifteen of thousands of known proteins that can be modified to include one or more unnatural amino acids, for example, by adapting any of the available mutation methods to include one or more appropriate selector codons in a relevant translation system. Common sequence depositories for known proteins include the GenBank EMBL, the DDBJ and the NCBI. Searching the internet can easily identify other depository files.

twenty Normally, proteins have an identity of, for example, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95% or at least 99% or more with any of the available proteins (for example, a therapeutic protein, a diagnostic protein, an industrial enzyme or a part thereof, and the like), and comprise one or more unnatural amino acids. Examples of therapeutic, diagnostic and protein proteins

25 Other proteins that can be modified to comprise one or more unnatural amino acids can be found, but not limited to, in International Publications WO 2004/094593, filed on April 16, 2004, entitled "EXPANDING THE EUKARYOTIC GENETIC CODE"; and WO 2002/085923, entitled "IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS". Examples of therapeutic, diagnostic and other proteins that can be modified to comprise one or more natural amino acids include, but are not

30 limitation, for example, hirudin, alpha-1 antitrypsin, angiostatin, antihemolytic factor, antibodies (more details on antibodies are found below), apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atrial peptides, CXC chemokines (for example, T39765, NAP-2, ENA-78, Gro-a, Gro-b, Gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG), calcitonin, CC chemokines (for example , monocyte chemoattractant protein-1, monocyte chemoattractant protein-2, monocyte chemoattractant protein-3,

35 Monocyte inflammatory alpha protein-1, Monocyte inflammatory beta protein-1, RANTES, I309, R83915, R91733, HCC1, T58847, D31065, T64262), CD40 ligand, C-kit ligand, collagen, colony stimulating factor (CSF) , Complement factor 5a, complement inhibitor, complement receptor 1, cytokines, (eg, epithelial neutrophil activator peptide 78, GROα / MGSA, GROβ, GROγ, MIP-1α, MIP-18, MCP-1), Factor of Epidermal Growth (EGF), Erythropoietin ("EPO"), exfoliating toxins A and B, Factor IX, Factor VII, Factor VIII,

40 X Factor, Fibroblast Growth Factor (FGF), Fibrinogen, Fibronectin, G-CSF, GM-CSF, glucocerebrosidase, gonadotropin, growth factors, hedgehog proteins (e.g., Sonic, Indian, Desert), hemoglobin, Growth Factor Hepatocyte (HGF), hirudin, human serum albumin, insulin, insulin-like growth factor (IGF), interferons (for example, IFN-α, IFN-β, IFN-γ), interleukins (for example IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, etc.), keratinocyte growth factor (KGF),

Four. Five lactoferrin, leukemia inhibitor factor, luciferase, neurturin, neutrophil inhibitor factor (NIF), oncostatin M, osteogenic protein, parathyroid hormone, PD-ECSF, PDGF, peptide hormones (for example, Human Growth Hormone), pleiotropin, Protein A , Protein G, Pyrogenic exotoxins A, B and C, relaxin, renin, SCF, Soluble complement receptor I, Soluble I-CAM 1, soluble interleukin receptors (IL-1, 2, 3, 4, 5, 6, 7 , 9, 10, 11, 12, 13, 14, 15), soluble TNF receptor, somatomedin, somatostatin, somatotropin, streptokinase,

fifty superantigens, ie 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 beta factor (TNF beta), tumor necrosis factor receptor (TNFR), tumor necrosis factor alpha (TNF alpha), Vascular Endothelium Growth Factor (VEGEF), urokinase and many others.

55 A class of proteins that can be prepared using the compositions and methods for in vivo incorporation of unnatural amino acids described herein include transcriptional modulators or a portion thereof. Examples of transcriptional modulators include genes and transcriptional modulating proteins that modulate growth, differentiation, cell regulation or the like. Transcriptional modulars are found in prokaryotes, viruses and eukaryotes that include fungi, vegetables, yeasts,

60 insects and animals including mammals, providing a wide range of therapeutic targets. It will be appreciated that expression and transcription activators regulate transcription by many mechanisms, for example, binding to receptors, stimulating a signal transduction cascade, regulating the expression of transcription factors, binding promoters and enhancers, joining proteins that bind to promoters and enhancers, unwinding the DNA, by splicing the pre-mRNA, polyadenylating the RNA and

65 degrading RNA.

A class of proteins of the present disclosure (for example, proteins with one or more unnatural amino acids) include biologically active proteins such as hirudin, cytokines, inflammatory molecules, growth factors, their receptors, and oncogenic products, for example, interleukins ( for example IL-1, IL-2, IL-8, etc.), interferons, FGF, IGF-I, IGF-II, FGF, PDGF, TNF, TGF-α, TGF-β, EGF, KGF, SCF / c-Kit, CD40L / CD40, VLA

5 4 / VCAM-1, ICAM-1 / LFA-1, and hyalurine / CD44; signal transduction molecules and corresponding oncogenic products, for example, Mos, Ras, Raf and Met; and transcription activators and suppressors, for example, p53, Tat, Fos, Myc, Jun, Myb, Rel and steroid hormone receptors such as estrogen, progesterone, testosterone, aldosterone, LDL receptor and corticosterone receptor ligands .

The description also provides enzymes (for example industrial enzymes) or parts thereof with at least one unnatural amino acid. Examples of enzymes include, but are not limited to, for example, amidases, amino acid racemases, acylases, dehalogenases, dioxygenases, diarylpropane peroxidases, epimerases, epoxide hydrolases, esterases, isomerases, kinases, glucose isomerases, glucosidases, glucosyl transferases, haloperoxidases (monooperoxases) for example p450s), lipases, lignin peroxidases, nitrile hydratases, nitrilases,

fifteen proteases, phosphatases, subtilisins, transaminases and nucleases.

Many of these proteins are commercially available (See, for example, the Sigma BioSciences 2002 catalog and price list) and the corresponding protein sequences and genes and, normally, many variants thereof, are well known (see , for example, Genbank). Any of them can be modified by inserting one or more unnatural amino acids according to the invention, for example, to alter the protein with respect to one or more therapeutic, diagnostic or enzymatic properties of interest. Examples of therapeutically relevant properties include serum half-life, conservation half-life, stability, immunogenicity, therapeutic activity, detectability (for example, by including indicator groups (e.g., markers or marker binding sites) in unnatural amino acids ), reduction

25 of LD50 or other side effects, ability to enter the body through the gastric tract (for example, oral availability) or the like. Examples of diagnostic properties include conservation half-life, stability, enzyme activity, production capacity or the like.

Using the compositions and methods of the invention, several different proteins can also be modified to include one or more unnatural amino acids. For example, the invention may include replacing one or more natural amino acids in one or more vaccine proteins with an unnatural amino acid, for example, in infectious fungal proteins, for example Aspergillus, Candida species; bacteria, particularly E. coli, which serves as a model of pathogenic bacteria, as well as medically important bacteria such as Staphylococci (for example aureus), or Streptococci (for example pneumoniae); protozoa such as sporozoa (for example

35 Plasmodia), rhizopods (for example Entamoeba) and flagellate (Trypanosoma, Leishmania, Trichomonas, Giardia, etc.); viruses such as RNA (+) viruses (examples include Poxvirus, for example, vaccinia; Picornavirus, for example, polio; Togavirus, for example, rubella; Flavivirus, for example, HCV; and Coronavirus), RNA virus (- ) (for example, Rabdovirus, for example, VSV; Paramyxovirus, for example, VSR; Ortomixovirus, for example, influenza; Bunyavirus; and Arenavirus), cDNA virus (for example, Reovirus), RNA to DNA virus, that is, Retrovirus, for example, HIV and VLTV and certain DNA to RNA viruses such as Hepatitis B virus.

Agriculture-related proteins, such as insect resistance proteins (for example Cry proteins), starch and lipid production enzymes, plant and insect toxins, toxin resistance proteins, mycotoxin detoxifying proteins, enzymes of plant growth (for example, Ribulosa 1.5

Four. Five Carboxylase / Oxygenase bisphosphate, "RUBISCO"), lipoxygenase (LOX) and Phosphoenolpyruvate (PEP) carboxylase are also appropriate targets for modification with unnatural amino acids.

In certain embodiments, the protein or polypeptide of interest (or parts thereof) in the methods and / or compositions of the invention are encoded by a nucleic acid. Typically, the nucleic acid comprises at least one selector codon, and may comprise at least two selector codons, at least three selector codons, at least four selector codons, at least five selector codons, at least six selector codons, at least seven selector codons , at least eight selector codons, at least nine selector codons, ten or more selector codons.

55 The genes encoding proteins or polypeptides of interest can be mutagenized using methods well known to those skilled in the art and described herein in "mutagenesis and other molecular biology techniques" to include, for example, one or more selector codons for incorporation of an unnatural amino acid. For example, a nucleic acid is mutagenized for a protein of interest to include one or more selector codons, providing the insertion of one or more unnatural amino acids. The invention includes any of said variants, for example, mutants, versions of any protein, for example, which includes at least one unnatural amino acid. Similarly, the invention also includes corresponding nucleic acids, that is, any nucleic acid with one or more selector codons encoding one or more unnatural amino acids.

65 To make a protein that includes an unnatural amino acid, cells and host organisms that are adapted for in vivo incorporation of the unnatural nucleic acid by orthogonal tRNA / RS pairs can be used. Host cells are engineered (for example, transformed, transduced or transfected) with one or more vectors that express orthogonal tRNA, orthogonal tRNA synthetase and a vector that encodes the protein to be derivatized. Each of these components may be in the same vector or each of them may be in a different vector, or the two components may be in a vector and the

5 third component in a second vector. The vector may be, for example, in the form of a plasmid, bacteria, virus, naked polynucleotide or conjugated polynucleotide.

Definition of polypeptides by immunoreactivity

10 As the polypeptides of the invention provide various new polypeptide sequences (for example, polypeptides comprising unnatural amino acids in the case of proteins synthesized in the translation systems of the present document or, for example, in the case of new synthetases, new sequences of conventional amino acids), polypeptides also provide new structural characteristics that can be recognized, for example, in immunological assays. The generation of antisera, which bind

fifteen specifically to the polypeptides of the invention, as well as the polypeptides that are bound by said antisera, constitutes a feature of the invention. The term "antibody", as used herein, includes, but is not limited to, a polypeptide substantially encoded by one or more immunoglobulin genes, or fragments thereof that specifically bind and recognize an analyte (antigen). Examples include polyclonal, monoclonal, chimeric, single chain antibodies and the like. The fragments of

twenty Immunoglobulins, including Fab fragments and fragments produced by an expression library, including phage display, are also included in the term "antibody" as used herein. See, for example, Paul, Fundamental Immunology, 4th ed., 1999, Raven Press, New York, for antibody structure and terminology.

25 In order to produce the antisera for use in an immunoassay, one or more immunogenic polypeptides are produced and purified as described herein. For example, the recombinant protein can be produced in a recombinant cell. An inbred mouse strain (used in this assay since the results are more reproducible due to the implicit genetic identity of the mice), is immunized with the immunogenic protein (or proteins) in combination with a conventional adjuvant, such as adjuvant of

30 Freund, and a conventional mouse immunization protocol (see, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a conventional description of antibody generation, immunoassay formats and conditions which can be used to determine specific immunoreactivity). Additional details on proteins, antibodies, antisera, etc. can be found. in International Publications Numbers WO 2004/094593, entitled “EXPANDING THE EUKARYOTIC

35 GENETIC CODE ”; WO 2002/085923, entitled "IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS"; WO 2004/035605, entitled "GLYCOPROTEIN SYNTHESIS"; and WO 2004/058946, entitled "PROTEIN ARRAYS".

USE OF O-tRNA AND O-RS AND O-tRNA / O-RS PAIRS

40 The compositions of the invention and the compositions prepared by the methods of the invention as defined in the claims are optionally in a cell. The O-tRNA / O-RS pairs or individual components of the invention that can then be used in a host system translation machinery, resulting in the incorporation of an unnatural amino acid into a protein. International Publication Number WO 2002/085923 by Schultz, et al., Entitled "IN VIVO INCORPORATION OF UNNATURAL

Four. Five AMINO ACID, ”describes this process. For example, when an O-tRNA / O-RS pair is introduced into a host, for example, Escherichia coli, the pair leads to the in vivo incorporation of an unnatural amino acid, which can be added exogenously to the culture medium, in a protein, for example, a myoglobin test protein

or a therapeutic protein, in response to a selector codon, for example, a codon without amber sense. Optionally, the compositions of the invention may be in an in vitro translation system, or in one or 50 more in vivo systems. Proteins with the unnatural amino acid can be used in any of a wide variety of applications. For example, the unnatural moiety incorporated in the protein can serve as a target for any of a wide variety of modifications, for example, cross-links with other proteins, with small molecules such as markers or dyes and / or biomolecules. With these modifications, the incorporation of the unnatural amino acid can result in improved therapeutic proteins and can

55 be used to alter or improve the catalytic function of enzymes. In some aspects, the incorporation and subsequent modification of an unnatural amino acid in a protein can facilitate studies on the structure of proteins, interactions with other proteins and the like.

Examples

60 The following examples are offered to illustrate, but not to limit, the claimed invention.

EXAMPLE 1

65 Genetic selection of sulfotyrosine-specific mutant synthetases

Methodologies that allow the systematic addition of unnatural amino acids to the genetic codes of E. coli cells (Wang et al., "Expanding the genetic code of Escherichia coli", Science 292: 498-500 (2001)), yeast ( Chin et al., "An expanded eukaryotic genetic code", Science 301: 964-967 (2003)) and mammalian (Zhang et al., "Selective incorporation of 5-hydroxytryptophan into proteins in mammalian cells", Proc Natl Acad Sci USA 101:

5 8882-8887 (2004)) have been described above. These methods are based on the development of an antisense suppressor tRNA / aaRS pair that has the property of orthogonality, defined as the ability to selectively incorporate a given amino acid in response to a single codon without reacting in cross-linking with tRNAs, aminoacyl- TRNA synthetases or the engendered amino acids of the host.

10 To generate an orthogonal tRNA / aaRS pair that only inserts sulfothyrosine (FIG. 1), a library of active site mutants of the tyrosyl-tRNA synthetase of Methanococcus jannaschii (MjTyrRS), which specifically loads a nonsense suppressor of M. jannaschii (MjARNtTyrCUA) modified by genetic engineering not recognized by E. coli synthetases (Wang et al., “Expanding the genetic code of Escherichia coli”, Science 292: 498-500 (2001)). This library, whose design and generation is described anywhere (Bose et al., “The

fifteen incorporation of a photoisomerizable amino acid into proteins in E. coli ”, J Am Chem Soc 128: 388-389 (2006)), underwent a series of positive and negative selections (3 positive and 2 negative). Survival in the positive selection depends on the suppression of an amber mutation in the chloramphenicol acetyltransferase (CAT) gene in the presence of 2 mM sulfothyrosine; Survival in the negative selection depends on the inadequate suppression of three amber mutations in a gene that encodes the toxic barnase protein in the absence of sulfothyrosine (Wang et al.,

twenty “Expanding the genetic code of Escherichia coli”, Science 292: 498-500 (2001)). Clones survive through both positive and negative selection rounds only if they incorporate only sulfothyrosine in response to the amber codon.

After these selections, numerous clones were identified that allowed the cells, which harbored the gene

25 CAT with an amber mutation at the permissive site 112, survive on chloramphenicol 130 μg / ml in the presence of 2 mM sulfothyrosine. In the absence of sulfothyrosine, the same cells did not grow in chloramphenicol 20 μg / ml, consistent with the efficient incorporation of sulfothyrosine with little to no background of incorporation of endogenous amino acids. Sequencing of the candidate mutant synthetase clones (called STyrRS) revealed four different synthetase clones, each of which met the criteria of an orthogonal translation system. He

30 Clone 1 was predominantly (Tyr32Leu, Leu65Pro, Asp158Gly, Ile159Cys, Leu162Lys). In FIG. 7 Nucleotide and amino acid sequences of each of these clones and wild-type species are provided.

<dl><dt>Mjtirosil-tRNA synthetase amino acid (and corresponding codon) </dt><dd /></dl>

<dl><dt>32 </dt><dd>65 155 158 159 162 SEQ ID NO: </dd></dl>

<dl><dt>wild type </dt><dd>Tyr (TAC) Leu (TTG) Gln (CAG) Asp (GAT) Ile (ATT) Leu (TTA) 2. 3) </dd></dl>

<dl><dt>Clone 1 </dt><dd>Leu (CTG) Pro (CCT) Gln (CAG) Gly (GGT) Cys (TGT) Lys (AAG) Four. Five) </dd></dl>

<dl><dt>Clone 2 </dt><dd>Leu (CTG) Pro (CCG) Gln (CAG) Gly (GGT) Thr (ACT) Lys (AAG) 6 (7) </dd></dl>

<dl><dt>Clone 3 </dt><dd>Leu (CTG) Pro (CCT) Glu (GAG) Gly (GGT) Cys (TGT) Lys (AAG) 8 (9) </dd></dl>

<dl><dt>Clone 4 </dt><dd>Leu (CTG) Pro (CCG) Gln (CAG) Gly (GGT) Ile (ATT) Lys (AAG) 10 (11) </dd></dl>

35 It is possible to assign possible functions of these mutations, particularly Lys162, which probably forms a salt bridge interaction with sulfotyrosine SO3-. Leu32 and Gly158 can house the larger SO3-group and eliminate the affinity of the endogenous tyrosine (Tyr32 and Asp158 are involved in the formation of hydrogen bonds with the tyrosine phenolic group in the wild-type enzyme). The replacement of anionic Asp158 with Gly possibly obvious unfavorable electrostatic interactions with sulfothyrosine. However, to perform

40 or using the invention, an understanding of the mechanism or functions of the various substituted positions is not required.

DETAILED METHODOLOGY FOR THE SELECTION OF SULFOTIROSINE AMINOACIL ARNt SYNTHEASE

Four. Five To select the STyrRS, an MjTyrRS active site library contained in the vector pBK (pBK-lib) (Bose et al., "The incorporation of a photoisomerizable amino acid into proteins in E. coli", J Am Chem Soc was used 128: 388389 (2006)). DH10B cells that included pRep, a positively selected plasmid containing a genetically engineered MjARNtTyrCUA, a chloramphenicol acetyltransferase gene with an amber codon introduced at position 112 (a permissive site) and a tetracycline resistance marker, were transformed with pBK-lib and seeded on agar plates with GMML supplemented with 2 mM sulfotyrosine (Senn Chemicals) and 68 μg / ml chloramphenicol. After 72 hours at 37 ° C, the plates scraped and the pBK-lib vectors were extracted.

5 This library plasmid collection was then used to transform DH10B cells containing pNeg, a negative selection plasmid, containing a genetically engineered MjRNAtTyrCUA, a toxic barnase gene with three amber codons introduced, and a chloramphenicol resistance marker . The cells were seeded on LB agar plates that did not contain sulfothyrosine and were grown at 37 ° C for 12 hours after which the pBK-lib vectors were extracted from the surviving cells. This positive and negative selection cycle was repeated once, and the selected pBK-lib vectors were subsequently transformed into DH10B cells containing pRep and replicated by plating on GMML agar plates with and without sulfotyrosine. Cells that grew on plates containing chloramphenicol 130 μg / ml in the presence of sulfothyrosine but did not grow on plaques containing chloramphenicol 20 μg / ml in the absence of sulfothyrosine were considered good successes.

fifteen These successes were classified and the orthogonality of their corresponding synthetases was confirmed by expressing the Z domain protein that contained an amber codon at position 7 in the presence and absence of sulfothyrosine. Orthogonal synthetases were those that allowed full length Z domain expression alone or in the presence of sulfothyrosine. MALDI-TOF was used to confirm that sulfotyrosine was actually incorporated into the full length Z domain.

EXAMPLE 2

Expression and characterization of a mutant model protein that contains sulfothyrosine

25 To verify the unique incorporation of sulfotyrosine by the selected STyrRS synthetase, an amber mutant (residue 7) of a His6-labeled Z domain protein was expressed in E. coli that included plasmids for the amber mutant Z domain , MjARNtTyrCUA and STyrRS (clone 1). Polyacrylamide gel electrophoresis (PAGE) analysis after purification with Ni-NTA showed a strong band for the Z domain only when the protein was expressed in media containing 2 mM sulfothyrosine - no band was observed in the absence of sulfothyrosine, which confirmed the dependence of amber suppression on sulfothyrosine (FIG. 4A).

For further characterization, MALDI-TOF analysis was performed on the purified mutant Z domain. It should be noted that MALDI-TOF and ESI analyzes of sulfated tyrosine proteins result in loss

35 partial sulfate, whose degree depends on the rigor of the conditions (22, 23). Therefore, mild conditions were used in positive ion mode with a matrix at moderate pH (2,4,6-trihydroxy-acetophenone), in which a predominant peak [M + H] of 7876 Da appeared (Mteoric = 7877, 5 Da) corresponding to the Z domain that contained a single sulfothyrosine and lacked methionine. A small peak (&lt;10%) [M + H] of 7798 Da (Mteoric = 7797.5 Da) was also observed, which is the result of the loss of sulfate during MALDI-TOF, leaving tyrosine (FIG. 4B ). Although these mass spectrometry data alone do not rule out the incorporation of background tyrosine by STyrRS, it can be done based on the PAGE gel analysis. Therefore, STyrRS incorporates only sulfothyrosine, which allows the recombinant expression of sulfated proteins in bacteria.

EXAMPLE 3

Expression of a sulfated model protein (hirudin) derived from a superior organism

It was examined whether this orthogonal system for the production of sulfated proteins could be used to generate a selectively sulphated native protein normally biosynthesized only in higher organisms. For this, the hirudin protein was selected, which is sulfated at the 63 position of tyrosine. Hirudin, secreted by the medicinal leech Hirudo medicinalis, is the strongest natural thrombin inhibitor, and its recombinant form is administered clinically as an anticoagulant. However, the recombinant expression of hirudin in E. coli and yeast used for commercial production of the drug produces the unsulfated form (desulfohirudin) due to the absence of sulfotransferases necessary in these organisms (Markwardt, “Hirudin as alternative anticoagulant

55 -a historical review, ”Semin Thromb Hemost 28, 405-414 (2002)). Although although desulfohirudin is an effective thrombin inhibitor, its affinity for human thrombin is at least an order of magnitude lower than that of sulfohirudin, which has a Ki of approximately 20 fM (Braun et al., “Use of site-directed mutagenesis to investigate the basis for the specificity of hirudin, ”Biochemistry 27, 6517-6522 (1988)).

To express sulfohirudin, the STyrRS gene (clone 1) was cloned into the pSup vector structure containing six copies of MjRNATyrCUA with optimized promoters (Ryu and Schultz, "Efficient incorporation of unnatural amino acids into proteins in Escherichia coli," Nat Methods 3 : 263-265 (2006)). The hirudin gene with an amber codon at position 63 and a gIII periplasmic signal sequence was synthesized and inserted into the pBAD vector. After cotransformation of E cells. coli DH10B with both plasmids, expression was performed in a shaker flask in

65 minimum liquid medium with glycerol (GMML) supplemented with 10 mM sulfothyrosine. Since hirudin is small, the direction within the periplasm results in secretion effectively; therefore, sulfohirudin was purified directly from the medium concentrated by FPLC using an anion exchange column Q Sepharose followed by size exclusion chromatography to yield a yield of 5 mg / l. For comparison, desulfohirudin with tyrosine encoded at position 63 was similarly expressed and purified with a yield of 12 mg / l.

5 DETAILED METHODOLOGY FOR THE CLONATION, EXPRESSION AND PURIFICATION OF SULFOHIRUDINE AND DESULFOHIRUDIN

The gene corresponding to [Leu1, Thr2] -63-desulfohirudin (commercially known as Lepirudin (Refludan®)) fused with a glasmic periplasmic signal sequence for secretion was synthesized by BlueHeron® with Expression Optimization. This gene was inserted into the pBAD vector (Invitrogen) to produce pBAD-Hirudin under the control of the araBAD promoter. Quickchange site directed mutagenesis (Stratagene) was used to introduce TAG at position 63 of the Lepirudin gene to produce pBAD-HirudinTAG for sulfohirudin expression.

fifteen The gene corresponding to the selected STirRS (clone 1) was inserted into the pSup vector between the PstI and NdeI sites under the control of the glnS promoter to produce pSup-STyrRS. The pSup-STyrRS also contained six copies of genetically engineered MjRNATyrCUA under the control of the proK promoter.

Electrocompetent DH10B cells cotransformed with pSup-STyrRS and pBAD-HirudinTAG were grown in GMML medium with 50 μg / ml ampicillin, 20 μg / ml chloramphenicol and 10 mM sulfotyrosine at 37 ° C. When the cells reached an OD600 of 0.6, L-arabinose was added at a final concentration of 0.2% to induce protein expression. The cells were cultured for an additional 24 hours at 37 ° C. The cells were sedimented and the media was concentrated using a cellular agitator device.

25 The concentrated media was dialyzed against water and applied to an anion exchange column (HiLoad 26/10 Q Sepharose, GE Healthcare) previously equilibrated with 50 mM Tris-HCl, 1 mM EDTA and 10 mM β-mercaptoethanol, pH 7, Four. The proteins were eluted with a linear NaCl gradient of 0.025 to 1 M. The peak fractions were analyzed by PAGE. Fractions from a main peak that eluted at 0.3 M NaCl were pooled together, concentrated, dialyzed against water and applied to gel filtration (Superdex 200 10/300 GL, GE Healthcare). The proteins were eluted with Tris buffered saline (25 mM Tris-HCl, 125 mM NaCl and 2 mM KCl, pH 7.6). The final sulfohirudin concentration was determined by titration against 1 nM human α-thrombin (Diapharma) using 50 μM of Boc-Asp thrombin fluorogenic substrate (OBzl) -Pro-Arg-MCA (Peptides International, Inc.) to measure the thrombin activity This implies a stoichiometric inhibition of 1: 1 thrombin by hirudin, which is valid at the concentrations used as stipulated by the close binding kinetics.

35 (Szedlacsek and Duggleby, "Kinetics of slow and tight-binding inhibitors," Methods Enzymol 249: 144-180 (1995)). Similar procedures were used to express, purify and quantify [Leu1, Thr2] -63-desulfohirudin.

EXAMPLE 4 OF REFERENCE

Characterization of a genetically encoded sulphated hirudin

The resulting hirudins described in the previous example were characterized by PAGE analysis and each one was presented as a single band. Sulfohirudin could be distinguished from desulfohirudin since the former migrates farther than the latter to offer a gel shift (FIG. 2). MALDI-TOF analysis showed the masses

Four. Five correct [M + H] of both sulfohirudin (7059 Da; Mteoric = 7059.5 Da) and desulfohirudin (6979 Da; Mteoric = 6979.5 Da) with two peaks in the case of sulfohirudin due to sulfate loss resulting in a secondary signal [M + H-80] (see FIG. 5).

To further verify that this second peak resulted exclusively from mass spectral analysis, two experiments were performed. First, the fact that the elution of sulfohirudin from the anion exchange column occurs at an ionic strength greater than 10% compared to the elution of desulfohirudin under the same gradient conditions, which would allow the complete separation of the two hirudins if they had been present simultaneously. (This was confirmed by adding sulfohirudin with desulfohirudin). Since no desulfohirudin peak was observed in anion exchange purification

55 of sulfohirudin, determined by the absence of a peak of desulfohirudin in the mass spectra of the corresponding eluted fractions, it was concluded that when sulfohirudin was expressed, desulfohirudin did not occur.

Second, a control expression was performed in which no sulfotyrosine was added. A subsequent MALDI-TOF analysis of the crude concentrated media containing a mixture of all segregated proteins showed only a peak [M + H] of 6578 Da corresponding to the truncated protein resulting from an alternative TAG behavior as a termination codon. (Mteoric = 6575 Da); no peak corresponding to the full length protein was observed (see FIG. 6A). This differs from the expression in the presence of sulfothyrosine in that in the mass spectra both truncated and full-length protein peaks are found at approximately 65 the same intensities (see FIG. 6B), which suggests a strict amber suppression dependence. on the presence of sulfothyrosine. From these two experiments, it is concluded that the signal [M + H-80] in

the MALDI-TOF of sulfohirudin is exclusively attributable to the cleavage of SO3- during mass spectrometry, confirming that STyrRS loads its tRNA exclusively with sulfotyrosine without observable tyrosine aminoacylation

5 It should be noted that similar intensities of the truncated and full-length protein peaks in the mass spectra of the raw sulfohirudin expression media combined with the fact that the desulfohirudin expression produces approximately twice the protein that Sulfohirudin expression under the same conditions suggests the suppression of approximately half of the translation events during sulfohirudin expression. It can therefore be deduced that double suppression in our system will produce approximately 75% truncated protein and 25% full length, assuming the absence of effects in the context of amber suppression. It is contemplated that the presence of the truncated protein is due to a low permeability of anionic sulfothyrosine in E. coli cells, resulting in a decreased population of amino acid-loaded MjRNAtTyrCUA. In fact, the expression of hirudin using the same system, but with the highly permeable p-acetyl phenylalanine and its corresponding mutant synthetase, results in the incorporation of p

fifteen Acetyl phenylalanine with undetectable truncated protein (data not shown). A prodrug strategy for administering sulfothyrosine can therefore eliminate the presence of truncated protein and increase yield.

EXAMPLE 5 OF REFERENCE

Characterization of the biological activity of genetically encoded sulfohirudin

To characterize the efficacy of sulfohirudin expressed as an anticoagulant, the kinetics of thrombin inhibition were determined using a fluorogenic enzyme assay based on the simple progress curve method previously described in the literature (Cha, “Tight-binding inhibitors-- III. A new approach for the

25 determination of competition between tight-binding inhibitors and substrates - inhibition of adenosine deaminase by coformycin, ”Biochem Pharmacol 25: 2695-2702 (1976); Komatsu et al., "CX-397, a novel recombinant hirudin analog having a hybrid sequence of hirudin variants-1 and -3," Biochem Biophys Res Commun 196: 773-779 (1993)). In this test, 100 pM sulfohirudin or desulfohirudin were mixed with 50 μM fluorogenic substrate to which human α-thrombin was added to initiate the reaction. The cleavage of fluorogenic substrate by thrombin, whose activity inhibits sulfohirudin and desulfohirudin at different degrees, results in a graph of fluorescence intensity over time (FIG. 3).

The exact concentrations of hirudin and sulfohirudin were determined by titration against thrombin in a concentration range in which a 1: 1 binding could be assumed. As a result of the narrow binding kinetics 35 suitable for hirudin (Stone and Hofsteenge, "Kinetics of the inhibition of thrombin by hirudin," Biochemistry 25: 4622-4628 (1986)), these experimental data were adjusted to equation 1, producing Ki, kon and koff values ​​after the treatment of the extracted constants. This analysis offered Ki values ​​for sulfohirudin and desulfohirudin of 26 fM and 307 fM respectively, according to bibliographic publications.

M-1

(17). As expected, the kon value for sulfohirudin (0.95 x 108s-1) was higher than that of desulfohirudin (0.38 x 108 M-1 s-1), while the koff value for sulfohirudin it was smaller (0.22 x 10-5 s-1) than that of desulfohirudin (1.18 x 10-5 s-1). The following table shows an average of these kinetic constants of thrombin inhibition derived from an adjustment of non-linear progress curves over at least 3 readings with typical deviations.

<dl><dt>Ki </dt><dd>kon x 10-8 (M-1 s -1) koff x 105 (s-1) </dd></dl>

<dl><dt>Sulfohirudin </dt><dd> 26 ± 9,8 0,95 ± 0,56 0,22 ± 0,06 </dd></dl>

<dl><dt>Desulfohirudin </dt><dd> 307 ± 72 0,38 ± 0,07 1,18 ± 0,45 </dd></dl>

Four. Five The advantage of the greater affinity of sulfohirudin over desulfohirudin should be especially pronounced in the concentration range of thrombin weakly bound by their respective Ki (Szedlacsek and Duggleby, "Kinetics of slow and tight-binding inhibitors," Methods Enzymol 249: 144-180 (1995)). Therefore it is interesting that the physiological baseline concentration of the steady state of active human thrombin is within this range (Velan and Chandler, “Effects of surgical trauma and cardiopulmonary bypass on active thrombin concentrations and the rate of thrombin inhibition in alive, ”Pathophysiol Haemost Thromb 33: 144156 (2003)), which suggests a possible evolutionary impulse for sulphation in native leech hirudin. This observation should serve as an orientation for possible therapeutic applications for genetically encoded desulfohirudin (described herein) on the non-sulphated recombinant form

55 predominant.

The cotraductional incorporation of sulfotyrosine into proteins should make possible the efficient expression of many more selectively sulfated proteins in E. coli including antibodies, chemokine receptor motifs and coagulation factors, thus facilitating structural-functional studies as well as therapeutic application. Sulfated protein practice. In addition, this in vivo strategy can be applied to the construction of sulfated antibody libraries and presentation of sulfated protein phages, promising pathways inaccessible by available methods of peptide synthesis, native chemical linkage and expressed protein binding. Alternatively, it should be possible to extend this strategy to the direct expression of sulfated tyrosine proteins in eukaryotic organisms.

DETAILED METHODOLOGY OF THE KINETIC CHARACTERIZATION OF HIRUDINE SPECIES

EXPRESSED

The release of Boc-Asp 7-amino-4-methylcoumarin (OBzl) -Pro-Arg-MCA 50 μM as a result of the activity of

10 thrombin was monitored by measuring the fluorescence intensity (excitation wavelength = 365 nm; emission wavelength = 450 nm) with a fluorescent plate reader (Molecular Devices SpectraMax Gemini). The enzymatic reaction was performed in triplicate and repeated three times in 96-well plates at 37 ° C in 50 mM Tris-HCl buffer, pH 7.8, containing 0.1% Polyethylene Glycol 6000 (Fluka), 100 mM NaCl, and HSA 250 μg / ml (Calbiochem). The substrate Michaelis constant under these conditions is 11.6 μM (Komatsu et al., "CX-397, a novel recombinant

fifteen hirudin analog having a hybrid sequence of hirudin variants-1 and -3, ”Biochem Biophys Res Commun 196: 773-779 (1993)).

The kinetic parameters of thrombin inhibition by sulfohirudin and desulfohirudin expressed were extracted from nonlinear adjustment of progress curves obtained at 40 pM of α-thrombin and 100 pM of sulfohirudin or desulfohirudin 20 using the simple progress curve method, such as It has been previously described (Komatsu et al., “CX-397, a novel recombinant hirudin analog having a hybrid sequence of hirudin variants-1 and -3,” Biochem Biophys Res Commun 196: 773-779 (1993)). According to the mechanism of competitive inhibition of close, slow hirudin binding, product formation can be described by equation 1 (Stone and Hofsteenge, "Kinetics of the inhibition of thrombin by hirudin," Biochemistry 25: 4622-4628 ( 1986); Cha, “Tight-binding inhibitors - III. A new

25 approach for the determination of competition between tight-binding inhibitors and substrates - inhibition of adenosine deaminase by coformycin, ”Biochem Pharmacol 25: 2695-2702 (1976)):

in which P is the amount of product formed in time t and v and v are the initial and steady state equilibrium reaction rates. In equation 1, vs, γ and λ can be described by the following expressions:

in which

and

Using these equations, Ki and kon values ​​were determined. The value of koff is the product of kon and Ki. The 45 non-linear regression settings were calculated using the GraphPad Prism program.