Method of obtaining an aprotinine analog

The present invention relates to a method for producing an aprotinin analogue used in the preparation of medicaments.

Aprotinin (also known as bovine pancreatic trypsin inhibitor) is a basic protein present in several bovine organs and tissues such as lymph nodes, pancreas, lungs, parotid gland, spleen and liver. It is a single chain 58-amino acid residue polypeptide with the following amino acid sequence:

Arg Pro Asp Phe Cys Leu Glu Pro Pro Tyr Thr Gly Pro Cys Lys Ala Arg Ile Ile Arg Tyr Phe Tyr Asn Ala Lys Ala Gly Leu Cys Glu Thr Phe Val Tyr Gly Gly Cly Arg Ala Lys Arg Asn Asn Phe Lys Cheese Ala Glu Asp Cys Met Arg Thr Cys Gly Gly Ala.

The amino acid chain is connected by three disulfide bridges formed between Cys (5) and Cys (55), Cys (4) and Cys (38) and Cys (30) and Cys (51) respectively.

The isoelectric point of aprotinin is very high (approximately pl 10.5). This is mainly due to the relatively high content of positive amino acids - lysine and arginine. The three-dimensional structure of the aprotinin molecule is very compact, which makes it very stable against denaturation at high temperatures or with acids, bases and organic solvents, or against proteolytic degradation (see B. Kassell, Meth. Enzym. 19,1970, pp. 844-852) .

Aprotinin is known to inhibit various serine proteases, including trypsin, chymotrypsin, plasmin and kallikrein, and is used to treat acute pancreatitis, various shock conditions, hemorrhage due to excessive fibrinolysis, and myocardial damage (see, for example, JE Trapnell et al., Brit. J. Surg. 61.1974, p. 177; J. McMichan et al., Circulatory shock 9, 1982, p. 107; LM Auer et al., Acta Neurochir. 49, 1979, p. 207; G. Sher, Am. J. Obstet. Gynecol, 129, 1977, pp. 164; and B. Schneider, Artzneim Forsch, 26, 1976, p. 1606). High-dose aprotinin administration significantly reduces blood loss associated with heart surgery, including cardiopulmonary bypass surgery (see, for example, BP Bidstrup et al., J. Thorac. Cardiovasc. Surg. 97, 1989, st. 364 -372; W. van Oeveren et al., Ann. Thorac. Surg. 44, 1987, pp. 640-645).

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Some analogues of aprotinin are known, e.g. from U.S. Patent No. 4,595,674, which discloses aprotinin analogs and derivatives in which Lys (15) is replaced by residues Gly, Ala, Val, Leu, Ile, Met, Arg, La-acid butter, L-norvaline, L-norleucine, rlpki / rlnnobinndif luln T ncamirse lKn-r-Eur.oi o jt ki ro notantnuru r 2 0 jC O Oj mon ima on οΙλπι a «fv \ tv_

J VII UU1UU111 jr 1UU Α ^^_1ΐνΐ11υθνΐ j 11 j. AjU1U | ZVJ01V1 Wpio J7UVVlilV ł j 111 _X S UJUłłlllU U11U1V ^ 1 U | Z1 in which the Lys (15) is replaced by the residues Arg, Val, Ile, Leu, Phe, Gly, Ser, Trp, Tyr or Ala, and of which Met (52) is also replaced by Glu, Val, Leu, Thr or Ser residues.

European Patent No. 370,592 discloses aprotinin analogues in which one or more amino acids at positions 15, 16, 17, 18, 34 and 52 are replaced with an amino acid residue. WO 89/011968 discloses a method for producing aprotinin or aprotinin analogues in yeast, and particularly discloses analogs lacking one or two amino acid residues at the N-terminus and in which the residue Lys (41) and / or Arg (42) is replaced with another amino acid, in particular the Ser residue.

European Patent No. 339 942 discloses aprotinin analogues in which one or more amino acids at positions 1,2, 12-19, 38,41 and 42 are deleted or replaced with another amino acid residue. In addition to the substitution of Met (52) according to European Patent No. 238 993 and the substitution of Lys (41) and / or Arg (42) according to Patent No. 89/01968, which is carried out to facilitate the production of aprotinin in E. coli and yeast, the amino acid substitutions described in these references lie mainly in the protease binding region of the aprotinin molecule, and their purpose is to change the protease inhibition profile of aprotinin.

As described in earlier publications, after intravenous injection of native aprotinin to animals or volunteers, the level of inhibitor in plasma decreases rather rapidly as a result of transition to extracellular fluid with subsequent accumulation in the kidneys (I. Trautschold et al. In K. Heinkel and H. Schon (ed.), Pathogenese, Diagnostik, Klinik und Therepie der Erkrankungen des Exokrinen Pankreas, Schattauer, Stuttgart, 1964, p. 289; E. Habermann et al., Med Welt 24 (29), 1973, p. 1163-1167 ; H. Fritz et al., Hoppe-Seylers Z. Physiol. Chem. 350, 1969, pp. 1541-1550; and H. Kaller r. Eur J. Drug Metab. Pharmacokin. 2, 1978, rU. 79-85). After filtration, aprotinin binds almost quantitatively to the membrane of the brush border of the proximal renal tubule cells. Aprotinin is then absorbed into micropinocytic vesicles and phagosomes, followed by very slow degradation in phagolisosomes. It has been suggested that this type of transport is representative of peptides in general (M. Just and E. Habermann, NavnynScmiedebergs Arch. Pharmacol. 280, 1973, pp. 161-176; M. Just, Navnyn-Scmiedebergs Arch. Pharmacol. 287, 1975, pp. 85-95.

Microscopic and histopathological studies after aprotinin administration reveal changes in renal tissue in rats, rabbits and dogs following injection of relatively high doses of aprotinin (Bayer, Trasyloł, Inhibitor of proteinase; E. Glaser et al. In: "Verhandlungen der Deutschen Gesellschaft fur Innere Medizin, 78. Congress, Bergmann, Munich, 1972, pp. 1612-1614). Observed nephrotoxicity of aprotinin (e.g. manifested in the form of lesions) can be attributed to the accumulation of aprotinin in the tubules of the kidneys closer to it. This nephrotoxicity makes aprotinin less suitable for clinical purposes, especially those requiring high doses of inhibitor (such as cardiopulmonary bypass surgery).

Therefore, it would be important to produce analogs of aprotinin with reduced nephrotoxicity compared to native aprotymin.

The invention relates to a method for producing an aprotinin analogue with reduced nephrotoxicity, wherein in order to provide a reduced net positive charge, at least one positive amino acid residue located outside the protease binding region is removed or replaced with a neutral or negative charge amino acid residue, and / or in which at least one negative charge amino acid residue is inserted or added, and / or in which at least one neutral amino acid residue is replaced with a negative charge amino acid residue, and / or in which, to provide reduced stability, one or more amino acid residues are deleted, added or replaced with one or more other amino acid residues.

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In this context, the term "reduced positive resultant charge" means a charge of analogs with a lower positive resultant charge than the native charge aprotinin (with a positive resultant charge of + 6), as well as without any accidental charge, or with a nipmnwn charge NalpT-ν 7anwa7vć 7P LOADNVNNV mnw rńżnip cip in TalpTnnćri nd ~ -j .. ^ -. w.,. pH and that the terms "positive net charge," negative net charge, "positive charge or" negative charge are used to charge the molecule at neutral pH.

By "protease binding site" is meant amino acid residues important for protease inhibition, i.e. amino acid residues that are in close contact with the protease by binding to amino acid residues at or near the active site of the enzyme. Currently, such residues are considered (and, in this context, referred to as) amino acid residues at positions 12-18 and 34-39 (see H. Fritz and G. Wunderer, Artzneim.-Forsch. 33 (1), 1983, p. . 484). Removal, insertion or replacement of amino acid residues outside the protease binding site is only beneficial in order to avoid substantial changes in the protease inhibition profile by the analog of the method of the invention compared to the inhibitory profile of native aprotinin.

It has been surprisingly found that aprotinin analogues with a reduced additional net charge have significantly lower nephrotoxicity than native aprotinin. One of the reasons for lower nephrotoxicity may be that aprotinin analogues with a lower additional net charge have reduced binding affinity to the proximal tubules (brush border membrane) so that they are more excreted in the urine. This explanation is consistent with the statement of H. Fritz et al., Op. cit. who report that chemically modified aprotinin derivatives (tetra- and pentamaleoyl derivatives) that are less basic than native aprotinin do not bind to the isolated brush limb fraction but are quantitatively excreted in the urine.

On the other hand, chemically modified aprotinin differs significantly from aprotinin and other native proteins in that it contains amino acid derivatives not found in any naturally occurring macromolecules. Therefore, it should not be excluded that the lack of accumulation in the kidneys of chemically modified derivatives can be attributed to other changed properties of the modified derivatives than the reduced positive net charge.

Other peptides have been found to bind to the brush limb with less affinity and efficiency than aprotinin, despite the content of positively charged amino acids and / or having a positive net charge. This indicates that the positive net charge charge of aprotinin is not the only explanation for its binding and accumulation in proximal tubular cells (M. Just et al., 1st. Synmp. Physiol., Prop. Pharmacol. Ration .: Kininogenases, Schattauer, Stuttgart 1973 , pp. 1163-1167).

In addition, it has surprisingly been found that aprotinin analogues with reduced thermal stability do not accumulate in renal tissue to the same extent as native aprotinin. As described above, the three-dimensional structure of aprotinin is very compact, which is believed to give the inhibitor high stability against denaturation and proteolytic degradation. Thus, accumulation of aprotinin in the kidneys may also be due to the exceptional stability of the inhibitor. It is possible because the result of substitution of one or more amino acid residues in the aprotinin molecule is reduced stability compared to the native molecule. In this context, "reduced stability can be expressed for selected purposes as reduced thermal stability of the analog in aqueous solutions at a pH of about 4-10. The in vivo impact of such reduced stability may be to make the analogue more susceptible to degradation, e.g. proteolytic degradation, which results in faster elimination of the analog from proximal tubules.

It is currently believed that the reduced nephrotoxicity of the aprotinin analogs produced by the method of the invention may result from a combination of reduced positive net charge and reduced stability of the molecule.

The reason contributing to kidney damage resulting from the administration of native aprotinin may be that it accumulates on the glomerular membranes due to the affinity for negatively charged structures on the membrane surface. This may result in increased glomerular pore size and, consequently, increased permeability of larger ones

168 250 molecules, for example albumin, which in turn can lead to kidney protein overload. It is likely that the aprotinin analog produced by the method of the invention may have, due to reduced affinity for negatively charged glomerular membrane structures, a smaller effect on the pore size of this membrane native native aprotinin.

In addition, it has been observed in some cases that administration of aprotinin leads to anaphylactoid response. The hypothesis has arisen that this anaphylactoid response is associated with the release of histamine, which may be triggered by a positive net charge of aprotinin. Therefore, it was assumed that the anaphylactoid response presumably associated with the administration of native aprotinin can be reduced or even eliminated by administration of the aprotinin analog produced by the method of the invention.

According to the invention, any of the positively charged amino acid residues outside the protease binding site can be replaced by either a negatively charged amino acid residue - Glu or Asp, or any of the neutral amino acid residues - Ala, Cys, Phe, Gly, His, Ile, Leu , Met, Asn, Pro, Glu, Ser, Thr, Val, Trp or Tyr. However, in order to avoid inactive analogs or analogs with an incorrect three-dimensional structure resulting from undesirable structuring of the molecule, it is preferable to select substituents that are identical to amino acid residues at the corresponding positions of other protease inhibitors, or in the domains of larger systems showing a high degree of homology to native apfotinin. In other words, the choice of substituent amino acid residue is preferably based on the analysis of aprotinin homologous molecules. It should be noted that simultaneously with the amino acid substitution (s) directly contributing to the reduction of the positive net charge, one or more substitutions of other amino acids can be made that do not themselves lower the positive net charge, but which may be required to produce an active analog with the appropriate structure three-dimensional.

A method for preparing the aprotinin analogue of the general formula IR¹ Asp Phe Cys Leu Glu Pro Pro R² Thr Gly Pro Cys Lys Ala Arg Ile Ile R³ Tyr Phe Tyr R⁴ Ala R⁵ Ala Gly Leu Cys R.⁶ Thr Phe Val Tyr Gly Gly Cys Arg R7 R⁸ R⁹ Asn R¹⁰ Phe R.¹¹ Ala Glu Asp Cys Met R cheese¹2 Thr Cys Gly Gly Ala, in which R1 is a dipeptide selected from the group consisting of Arg-Pro, Glu-Pro, Asp-Pro, Ala-Pro, Ile-Pro, Thr-Pro, His-Pro, Leu-Pro, Gly- Pro and Ser-Pro, Pro or R¹ is hydrogen, R2 is an amino acid residue selected from the group consisting of Tyr, Glu, Asp, Ser, Thr, Ala and Val, R3 is an amino acid residue selected from the group consisting of Arg, Glu, Asp, Leu, Ser, Ala, Gin and Thr, R4 is the amino acid residue selected from the group consisting of Asn, Glu and Asp, R5 is the 'amino acid residue selected from the group consisting of Lys, Glu, Asp, Thi, Val, Ala, Ser, Phe, Gin and Gly, R6 is the residue amino acid selected from the group consisting of Gin, Glu, Asp, Val and Ala, R7 is an amino acid residue selected from the group consisting of Ala, Asp, Glu and Gly, R⁸ is an amino acid residue selected from the group consisting of Lys, Glu, Asp, Asn, Ser, Thr and Ala, R9 is an amino acid residue selected from the group consisting of Arg, Glu, Asp, Ser, Asn, Leu, Gly, Gin, Met 'and Thr, R "means an amino acid residue selected from the group consisting of Asn, Glu and Asp, R" means an amino acid residue selected from the group consisting of Lys, Glu, Asp, Leu, Tyr, Ala, Val, Thr, Ser, Pro, His and Ile and R12 is an amino acid residue selected from the group consisting of Arg, Glu, Asp, Gin, Ala, Asn, His, Gly, Ser and Thr, except that at least one of the amino acid residues R "to R" is different from the corresponding amino acid residue of native aprotinin, and that when R "is hydrogen, then at least one of the amino acid residues R2 to R" is other than the corresponding amino acid residue of native aprotinin, except for aprotinin analogs 3-58 and aprotinin analogues (3-58, 42 Ser), according to the invention, that the cell containing the recombinant expression vector constituting the DNA construct containing the DNA sequence encoding this aprotinin analog is cultured under conditions that allow expression of the aprotinin analog and the resulting analog is recovered from the culture.

Preferably, a DNA construct comprising a DNA sequence encoding an aprotinin analogue, comprising a DNA sequence encoding one or more negative or neutral charge amino acid residues added at the 5 'end of the aprotinin coding sequence is preferably used.

Preferably, a DNA construct comprising a DNA sequence encoding an aprotinin analogue also comprising a DNA sequence encoding one or more negative or neutral charge amino acid residues added at the 3 'end of the aprotinin coding sequence is also preferably used.

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In addition to internal substitutions in the aprotinin molecule, it is possible to add a peptide containing one or more negative charge amino acid residues (i.e., Glu or Asp) at the N- or C-terminus of the aprotinin molecule, to provide the required reduction in the positive charge of UlVgV ΚΛ-νΐΙΛΙΑΙλΙΑ ιιχνι.

it is possible to add one or more neutral amino acid residues at the N- or C-terminus of the plate. Such attachments can be made either to the native aprotinin molecule or to other modifications as indicated above.

If it is desired to change the inhibitory properties of the aprotinin analogue proteases, in addition to reducing its nephrotoxicity, additional analogue modifications at the protease binding site are possible. For example, previously performed (see HR Wenzel and H. Tschesche, Angew. Chem. Internat. Ed. 20, 1981, p. 295) that aprotinin (1-58, Vall5) has relatively high specificity for granulocyte elastase and the inhibitory effect on collagenase, aprotinin (1-58, Ala15) has a weak effect on elastase, and aprotinin (1-58, Gly 15) shows high antitrypsin activity and, unexpectedly, also inhibits kallikrein. In addition, it is also possible to modify the inhibitory effect of aprotinin, simultaneously with a decrease in the positive net charge, by replacing one or more amino acids with a positive charge at the protease binding site with a neutral or negative amino acid (s).

Preferably, a DNA construct comprising a DNA sequence encoding an aprotinin analogue of the general formula IIR "Asp Phe Cys Leu Glu Pro Pro R2 Thr Gly Pro Cys R" Ri4 r "Ri6 Ri7 R³ Tyr Phe Tyr R4 Ala R⁵ Ala Gly Leu Cys R6 Thr Phe R "Tyr R" 9 Gly Cys r2 ° r7 R⁸ r9 Asn r1 ° Phe R "¹ Ser Ala Glu Asp Cys Met R "2 Thr Cys Gly Gly Ala, in which R" is a dipeptide selected from the group consisting of Arg-Pro, Glu-Pro, Asp-Pro, Ala-Pro, Ile-Pro, Thr-Pro, His-Pro, Leu-Pro, Gly-Pro and Ser-Pro, Pro or R "is a hydrogen atom, R2 is an amino acid residue selected from the group consisting of Tyr, Glu, Asp, Ser, Thr, Ala and Val, R3 is a residue amino acid selected from the group consisting of Arg, Glu, Asp, Leu, Ser, Ala, Gln and Thr, R4 is the amino acid residue selected from the group consisting of Asn, Glu and Asp, R⁵ is an amino acid residue selected from the group consisting of Lys, Glu, Asp, Thr, Val, Ala, Ser, Phe, Gln and Gly, R6 is an amino acid residue selected from the group consisting of Gln, Glu, Asp, Val and Ala, R7 is a residue amino acid selected from the group consisting of Ala, Asp, Glu and Gly, R⁸ is an amino acid residue selected from the group consisting of Lys, Glu, Asp, Asn, Ser, Thr and Ala, R9 is an amino acid residue selected from the group consisting of Arg, Glu, Asp, Ser, Asn, Leu, Gly, Gln, Met and Thr , R¹° means an amino acid residue selected from the group consisting of Asn, Glu and Asp, R "means an amino acid residue selected from the group consisting of Lys, Glu, Asp, Leu, Tyr, Ala, Val, Thr, Ser, Pro, His and Ile, and R "is an amino acid residue selected from the group consisting of Arg, Glu, Asp, Gln, Ala, Asn, His, Gly, Ser and Thr, except that at least one of the amino acid residues R" to R "is different from the corresponding one amino acid residue of native aprotinin and that when R "is hydrogen, then at least one of the amino acid residues R2 to R "2 is different from the corresponding amino acid residue of native aprotinin except that when R" is hydrogen, then R9 is other than Ser, R. " means an amino acid residue selected from the group consisting of Lys, Arg, Glu, Leu, Met, Tyr and Phe, R "* means an amino acid residue taken from the group consisting of Ala and Gly, Ri5 means an amino acid residue selected from the group consisting of Arg, Ala, Gly, Lys, Leu, Met, Phe, Tyr, Ile and As, R¹6 means an amino acid residue selected from the group consisting of Ile, Met, Leu, Phe, Thr and Glu, Ri7 means an amino acid residue selected from the group consisting of Ile, Leu, Lys, Gln, Glu, Ser, Arg, Thr and Asn, R "means amino acid residue selected from the group consisting of Val, Thr, Leu, Ser, Tyr, Gln, His, Pro, Phe, Asn, Ile and Lys, R "means the amino acid residue selected from the group consisting of Gly, Thr and Ser, and R2 ° means an amino acid residue selected from the group consisting of Gly, Lys, Met, Asn, Leu, Gly, Glu, with that at least one of the amino acid residues R to R and at least one of the amino acid residues R 3 to R 2 ° is different from the corresponding amino acid residue of native aprotinin, and that when R "is hydrogen, then R" has a different meaning than Ala, R "is me than Glu, and R9 is other than Ser and that when R" 5 is Ala, then R9 is me than Ser, except for aprotinin (3-58, 17Ala), aprotinin (3-58, 17Ala + 19Glu) , aprotinins (3-58, 15Arg + 17Ala), aprotinins (3-58, 17Ala-42Ser), aprotinin (3-58, 17Ala + 19Glu + 42Ser), aprotinin (3-58, 15Arg + 17Ala + 42Ser), aprotinin (17Ala + 4-2Ser) and aprotinin (15Arg + 17Ala + 42Ser).

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Examples of preferred DNA constructs used are constructs comprising DNA sequences encoding an aprotinin analogue of general formula (I) in which R¹ stands for Glu-Pro, R⁵is Glu, R8 is Glu, R is Glu, and R2, R³, R⁴, R⁶, r7, Rⁱⁿ and R1² are like in. and · lo τ · »1 z—, and τ» τύ 9 i ta 1 1 _ zi i _ »> 2 of the naive aprotinin sequence, luo r means Glu-Pro, r means Giu, r means Glu, ar,

R³, R4, R⁶, R ', R8, r1 and R12 have the same meaning as in the native sequence of aprotinin; or wherein R1 is Glu-Pro, R⁹ is Glu, R5 is Glu, and R1, R2, R³, r4, r5, r6, r7, r8, ro and R12 have the same meaning as in the native sequence aprotinin; or wherein R2 is Ser,

R4 is Asp, R5 is Thr, R6 is Glu, R⁸ is Asn, R12 is Glu, and R1, R³, R7,

R9, R1 ° and R11 have the same meaning as in the native sequence of aprotinin; or in which R2 is Ser, R is Leu, R is Gly, R is Asn, R is Gly, R is Gin,

R11 is Tyr, and R1, R4, r5, r6 and r2 have the same meanings as in the native aprotinin sequence;

or wherein R1 is hydrogen, R9 is Ser, R is Glu, and R2, R³, r4, r5, r6, r7,

R⁸, R¹⁰ and R12 have the same meanings as in the native sequence of aprotinin; or wherein R1 is hydrogen, R9 is Ser, R is Ala, and R2, R³, r4, R⁵, r6, r7, r8, rio and r12 _mhave the same meaning as in the native sequence of aprotinin; or wherein R is hydrogen,

R is Ser, R is Asp, R is Thr, R is Glu, R is Asn, R is

Glu, aR³, R⁷, R9, Ri^ABOUTiRii have the same meanings as in the native sequence of aprotinin; or wherein R is hydrogen, R4 is Asp, R⁵ is Thr, R6 is Glu, Ri2 is

Glu, aR, R, R, R, R, R and R have the same meaning as in the native sequence of aprotinin;

2 Or in which R is hydrogen, R is Ser, R is Gly, R is Asn, R

3 4 5 0 10 11 is Gly, R is Glu, and R, R, R, R, R and R have the same meaning as in the native sequence of aprotinin; or wherein R is hydrogen, R9 is Ser, R is Glu, and

R4, R³, R6, R7 'r8, rio and ^ Rii have the same meanings as in the native sequence

9 12 23 aprotinins; or wherein R is hydrogen, R is Glu, R is Glu, and R, R,

R2, R.³

R4, R⁵, r6, r7, r8, r1 ° and Ri1 have the same meaning as in the native sequence of aprotinin; or wherein R is hydrogen, R5 is Glu, R9 is Ser, R is Glu, and R2, r3, r4, R, IR, RR and R 'have the same meanings as in the native aprotinin sequence; or wherein R is hydrogen, R is Glu, R is Glu, R is Glu, and R, R, R, R, R, R8, Rio and Ri have the same meanings as in the native aprotinin sequence.

Amino acid sequences of the aprotinin analogues obtained according to the invention defined in sequences with identification numbers 2,4,6,8,10,12,14,16, 18, 20, 22, 24, 2, respectively (61 28. Sequence number 29 defines the amino acid composition of intravenous aprotinin.

The method of the invention uses a DNA construct encoding the analogs produced by the method of the invention. The DNA sequences encoding these aprotinin analogues with the abovementioned identification numbers present the sequences with the identification numbers 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27, respectively. can be prepared synthetically by known standard methods, for example the phosphoramidite method, described by SL Beauoage and MH Caruthers, Tetrahedron Letters 22, 1981, pp. 1859-1869, or by the method described by 'Matthes et al. EMBO Journal. 3, 1984, pp. 801-805. According to the phosphoramidite method, oligonucleotides are synthesized, for example in an automated DNA synthesizer, purified, combined, ligated and cloned into appropriate vectors.

Alternatively, genomic DNA or cDNA encoding native aprotinma (e.g. obtained by screening a genomic or cDNA library using synthetic oligonucleotide probes) and modification at one or more sites corresponding to the site (s) in which amino acid substitutions are desirable may also be used. for example, by site-directed mutagenesis using synthetic oligonucleotides encoding the desired amino acid sequence for homologous recombination, according to well known procedures.

The method of the invention also uses recombinant expression vectors containing the aforementioned DNA construction. The recombinant expression vector can be any vector that can be conveniently subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.

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Thus, the vector may be an automatically replicable vector, i.e. a vector that occurs as an extrachromosomal unit whose replication is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be a vector that, when introduced into the host cell, is integrated into its genome and replicated with the chromosome (s) with which it has been integrated.

The DNA sequence coding for the aprotinin analog produced by the method of the invention should be linked in a vector to allow operation with the appropriate promoter sequence. The promoter can be any DNA sequence that has transcriptional activity in a selected host cell and can be obtained from genes encoding proteins either homologous or heterologous to the host cell. Examples of promoters suitable for directing the transcription of DNA encoding the aprotinin analog produced by the method of the invention in mammalian cells are the SV 40 promoter (Subramiiin., Mol. CellBiol. 1,1981, pp. 854-864), the MT-1 promoter (metallothionein gene) (Palmiter et al., Science 222.1983, pp. 809-814 or major adenovirus 2 promoter late. Promoters suitable for use in yeast host cells include promoters of yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255.1980, pp. 12073-12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1,1982, pp. 419-434) or genes of alcohol dehydrogenases (Young et al., Genetic Engineering of Microorganisms for Chemicals (ed. Hollaender et al.), Plenum Press, New York, 1982), or promoters TPIi (US Patent No. 4,599,331) or ADH2-4c (Russell et al., Nature 304, 1983, pp. 652-654). Suitable promoters for use in filamentous fungal cells are e.g. the ADH3 promoter (McKnight et al., The EMBO J. 4, "985, pp. 2093-2099) or the tpiA promoter.

The DNA sequence encoding the aprotinin analog produced by the method of the invention can also be operably linked to a suitable terminator, such as the human growth hormone gene terminator (Palmiter et al., Op. Cit.) Or (for fungal hosts) TPIi promoters (Alber and Kawasaki , op. cit.) or ADH3 (McKnight et al., op. cit.). The vectors may further include elements such as polyadenylation signals (e.g. from SV 40 or Adenovirus Elb region), transcription enhancers (e.g. SV 40 enhancer) and translational enhancers (e.g. encoding adenovirus VA RNA).

The recombinant expression vector used in the method of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences (when the host cell is a mammalian cell) is the SV 40 origin of replication or (when the host cell is a yeast cell) REP 1-3 replication genes and plasmid 2 / j origin of replication. The vector may also contain a selection marker, e.g. a gene whose product complements a host cell defect, such as a gene encoding dihydrofolate reductase (DHFR), or a gene that confers resistance to a drug, e.g. neomycin, hygromycin or methotexate, or the TPI Schizosaccharomyces pombe gene (described by Rusel's PR, Gene 40, 1985, pp. 25-130.

The procedures used to ligate DNA sequences encoding respectively the aprotinin analog produced by the method of the invention, the promoter and terminator, and to incorporate them into appropriate vectors containing the information necessary for replication are well known to those skilled in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989).

The host cell into which the expression vector used in the method of the invention is introduced can be any cell that is capable of producing an aprotinin analog produced by the method of the invention and is preferably a eukaryotic cell, such as a mammalian cell, yeast cell or fungal cell.

The yeast organism used as the host cell can be any yeast organism that produces large amounts of the aprotinin analog during cultivation according to the invention. Examples of suitable yeast organisms are strains of the yeast species Saccharomyces cerevisia, Saccharomyces kluyveri, Schizosaccharomyces pombe and Saccharomyces uvarum. Transformation of yeast cells can, e.g., be achieved by forming protoplasts followed by transformation in a manner known per se.

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Examples of suitable mammalian cell lines are COS (ATCC CRL 1650), BHK (ATCC CRL 1632, ATTC CCL 10) or CHO (ATCC CCL 61) cell lines. Methods of transfection of mammalian cells and expression of DNA sequences introduced into cells are described, for example, in the public • -_ιτλ z * · m. τ ^ λ · · _ιτ. ....................-. . _ _ _. ____ cjdcn kiiuirndu and auaip. j. ινιυι. mui. uy, hi. oui-όζι, duuuicm and ncig. j. ινιυι. ^ ^ Ppi jcnci.

and, 1982, pp. 327-341; Loyter et al., Proc. Natl. Acad. Sci. USA 79.1982, pp. 422-426; Wigier et al., Cell 14,1978, p. 725; Corsaro and Pearson, Somatic Cell Genetics 7.1981, p. 603; Graham and van der Eb, Virology 52, 1973, pp. 456 and Neuman et al., EMBO J. i, 1982, pp. 841-845.

Alternatively, fungal cells can be used as host cells in the method of the invention. Examples of suitable fungal cells are filamentous fungal cells, e.g. Aspergillus spp. Or Neurospora spp., Especially strains of Aspergillus oryzae or Aspergillus niger. The use of Aspergillus spp. For the expression of proteins is described, for example, in European Patent No. 272277.

The medium used to grow the cells may be any conventional medium suitable for growing mammalian cells or yeast, depending on the choice of host cells. The aprotinin analog will be secreted by the cells into the growth medium and can be recovered therefrom in a known manner, including separation of the cells from the medium by centrifugation or filtration, precipitation of the protein components of the supernatant or salt filtrate, e.g. ammonium sulfate, purification by various chromatographic techniques, e.g., ion exchange or affinity chromatography, or the like.

The aprotinin analogs produced by the method of the invention find use in the preparation of pharmaceutical compositions containing the aprotinin analog produced by the method of the invention together with a pharmaceutically acceptable carrier or excipient. In such a composition, an aprotinin analog can be formulated using any of the known methods of formulating pharmaceutical compositions, e.g., as described in Remington's Pharmaceutical Sciences, 1985. The composition may typically be in a form suitable for systemic injection or infusion and, as such, may be formulated with sterile water or isotonic saline or glucose solution.

In addition, the aprotinin analog produced by the method of the invention can be used to produce a drug with reduced, compared to native aprotinin, nephrotoxicity and / or a drug which, when administered, causes fewer cases of anaphylactoid reactions than those found with native aprotinin.

As mentioned previously, it was found that native aprotinin, administered at doses close to the clinical doses, has harmful effects on the kidneys. This effect may be due to the unusually high stability and relatively high positive net charge charge of the aprotinin molecule. Therefore, the aprotinin analog produced by the method of the invention appears to be beneficial for use in therapeutic applications suggested for native aprotinin, especially those requiring high doses. Therapeutic applications for which the use of the aprotinin analog produced according to the invention is indicated by its inhibition of human serine proteases, e.g. trypsins, plasmin, kallikrein, elastases and cathepsin G, include (but are not limited to acute pancreatitis, inflammation, thrombocytopenia, platelet function protection, organ protection, wound healing, shock (including pulmonary shock) and conditions including hemorrhage caused by excessive fibrinolysis. A high dose of aprotinin is indicated during and after cardiopulmonary surgery involving bypassing the blood vessel; therefore, for this application, and perhaps for other operations that are accompanied by significant blood loss, the lower nephrotoxicity of the aprotinin analog produced by the method of the invention, and perhaps the reduced risk of anaphylactoid response due to the lower positive charge, the resultant analogue, is particularly important.

The method of the invention is illustrated in the following examples and drawing, in which Figure 1 shows the construction of a synthetic aprotinin gene from oligonucleotide sequences; Figure 2 - construction of plasmid pKFN-1503; Figure 3 is a bar graph illustrating the inhibitory activity in urine and kidneys after administration of aprotinin analogues with different net charge and thermal stability; FIG. 4 - inhibitory activity in urine 3 hours after administration of aprotinin analogues with different accident loads; Figure 5 - i2 inhibitory activity

OóS 25 ° in urine 3 hours after administration of aprotinin analogues with different thermal stability; and Figure 6 - accumulation of inhibitory activity in the kidney after administration of aprotinin analogues with different thermal stability. The accumulation index is calculated as the inhibitory activity νχ / Ά τ * «« - »//! ·»

11ŁW11 ^ VUĆj111UV11 .......

pXXVX XXXV xj »ππνον 1ΧΧΧΧΧΧ Vxjx. | VXX 1 ^ KZVXZ.iXXXX V ·.

Example 1. Preparation of (Glu1, Glu26, Glu41, Glu46) -aprotinin in the yeast strain KFN-1512.

From 10 oligonucleotides, a synthetic coding gene (Glul, Glu26, Glu41, Glu46) -aprotinm was synthesized by ligation. Oligonucleotides were synthesized in an automatic synthesizer, using the phosphinamide method, on a controlled pore size glass support (Beaucage, SL and Caruthers, MH, Tetrahedron Letters 22, (1981) 1859-1869).

The following 10 oligonucleotides were synthesized:

NOR-1948; CATGGCTGAGATTGGAGGAGAGAGCCTGATTTATGTTTGGAACCTC CATACACTGGTCC

NOR-1947: TTACATGGACCAGTGTATGGAGGTTCCAAACAGAAATCAGGCTCTCTCTT CTCCAATCTCTCAGC

TOR-354: ATGTAAAGCTAGAATCATCAGATACTTCTACAACG

NOR-193 9: MCGGCGTTGTAGAAGTATCTGATGATTCTAGCT

NOR-1938: CCGAAGCTGGTTTGTGTCAAACTTTCGTTTACGGTGGCT

NOR-357: CTCTGCAGCCACCGTAAACGAAAGTTTGACACAAACCAGC

NOR-194O: GCAGAGCTGAAAGAAACAACTTCGAAT

NOR-194 9: AGCAGATTCGAAGTTGTTTCTTTCAG

NOR -360: CTGCTGAAGAGTGCATGAGAACTTGTGGTGGTGCCTAAT

NOR-361: CTAGATTAGGCACCACCACAAGTTCTCATGCAGTCTTC

Five A-E duplexes were formed from the above 10 oligonucleotides, as shown in Figure 1.

pmoles of each of the A-E duplexes were formed from the corresponding oligunucleotide pairs with 5 'phosphorylated ends by heating for 5 minutes at 90 ° C, followed by cooling to room temperature over a period of 75 minutes. 5 duplexes were mixed and treated with T4 DNA ligase. After electrophoresis of the ligation mixture on a 2% agarose gel, the synthetic gene was isolated as a band corresponding to a length of 203 base pairs. The resulting synthetic gene is shown in Figure 1.

The synthetic gene was ligated with a 209 bp EcoRI-Ncol fragment of the pLaC212spx3 plasmid and a 2.8 kb EcoRI-XbaI fragment of the pTZi9R plasmid (Mead, DA

168 250 13

Szczesna-Skorupa, E. and Kemper, B., Prot. Engin. 1 (1986) 67-74). Plasmid pLaC212spx3 is described in Example III of International Patent Application No. PCT / DK88 / ÓX) 147.

A 209 bp EcoRI-NcoI fragment from plasmid pLaC212spx3 encodes a synthetic yeast tider peptide.

The ligation mixture was used to transform the competent E. coii strain (f^1-, m1⁺) using ampicillin resistance selection. DNA sequencing (Sanger, F., Micklen, S. and Coulson, AR, Proc. Natl. Acad. Sci. USA 74 () 777) 54635467) showed that the plasmids from the resulting colonies contained the correct DNA sequence [Glul, Glu26, Glu41 , Glu46] -aprotinin.

One plasmid, pKFN-1503 was selected for further use.

pKFN-1503 was digested with EcoRI and XbaI restrictionases and the 412 bp fragment was ligated to the 9.5 kb NcoI-XbaI fragment of the pMT636 plasmid and the 1.4 kb Ncol-EcoRI fragment to the pMY636 plasmid, resulting in plasmid pKFN-1508 , see Figure 3. The plasmid pMT636 is described in International Patent Application No. PCT / DK88 / (X) 138.

pMT636 is an E. coli shuttle vector - S. ^ κνϋδΐ »^ containing the TPI gene Schizosaccharomyces pombe (POT) (Russell, PR, Gene 40 (1985) 125-130), the promoter and terminator of the S. crrrvisiαr triosophosphate isomerase, TPIp and TPIt ( Alber, T. and Kawasaki, GJ Mol. Appl. Gen. i (1982), 419-434). Plasmid pKFN-1508 contains the following sequence:

TPIp-LaC212spx3 (1-47) Glu signal-leader sequence (ArgLeuGluLysArg [Glul, Glu26, Glu41, Glu46] -apTotininTPlT, where the LaC212spx3 signal-leader sequence is a synthetic yeast leader, described in international patent application No. 00 / PCT DK7. The DNA sequence of the 412 bp EcoRI-XbaI fragment of plasmids pKFN-1503 and pKFN-1508 is provided under the identification number and sequence list.

The S. cCTmsiae strain MT663 (E2-7B XEll-36 a / a, tpi / tpi, pep 4-3 / pep 4-3) was grown on YPGal (1% Bacto yeast extract, 2% Bacto peptone, 2% galactose, 1 % lactate) up to OD 0.6 at 600 nm.

100 ml of culture was collected by centrifugation, washed with 10 ml of water, centrifuged again and suspended in 10 ml of a solution containing 1.2 M sorbitol, 25 mM NagEDTA, pH 8.0 and 6.7 mg / ml dithiothreitol. The suspension was incubated at 30 ° C for 15 minutes, centrifuged and the cells resuspended in 10 ml buffer containing 1.2 M sorbitol, 10 mM Na2EDTA, 0.1 M sodium citrate, pH 8.5 and 2 mg Novozym® 234. The suspension was incubated at 30 ° C for 30 minutes, the cells were collected by centrifugation, washed with Wml 1.2M sorbitol and 10ml CAS (1.2M sorbitol, 10 mM CaCl2, 10 mM Tris HCl (Tris = Tris (hydroxymethyl) aminomethane), pH 7.5) and resuspended in 2 ml CAS. For transformation, 0.1 ml of cells resuspended in CAS were mixed with about 1 µg of plasmid pKFN-1508 and left at room temperature for 15 minutes. 1 ml of a solution containing 20% ​​polyethylene glycol 4000, 20 mM CaCl2, 10 mM CaCb, 10 mM Tris HCl, pH 7.5) was added and the mixture was left for a further 30 minutes at room temperature. The mixture was centrifuged and the pellet was resuspended in 0.1 ml SOS (1.2 M sorbitol, 33%, v / v YPD, 6.7 mM CaCle), 14pg / ml leucine) and incubated at 30 ° C for 2 hours. The suspension was then centrifuged and the pellet resuspended in 0.5 ml 1.2 M sorbitol. Then 6 ml of surface agar (SC medium according to Sherman et al., (Methods in Yeast Genetics, Cold Spring Harbor Laboratory (1982)) containing 1.2 M soTbitol plus 2.5% agar was added at 52 ° C. poured onto the surface of plates containing the same, agar, medium containing soTbitol.

Transformed colonies were transplanted after three days at 30 ° C, re-isolated and used to establish liquid cultures. One such transformant, KFN-1512, was selected for further characterization.

Yeast strain KFN-1512 was cultured on YPD medium (1% yeast extract, 2% peptone (Difco Laboratories) 6% glucose). 200 ml of the strain culture were shaken at 250 rpm at 30 ° C for 3 days, until an OD of about 20 was reached at 600 nm. After centrifugation, the supernatant was analyzed by FPLC ion exchange chromatography. Yeast supernatant. filtered through a Millex GV filter unit with a pore diameter of 0.22pm, and 1ml was applied to a cation exchange column. MonoS (0.5X5 cm) equilibrated with 20 mM formic acid, pH 3.7. After washing with equilibration buffer, the column was eluted with a linear NaCl gradient (0.1 M) in equilibration buffer. In eluted

168 250 fractions were spectrophotometrically determined for trypsin inhibitor activity (Kassel, B., Methods Enzymol. 19 (1970), 844-852), and the absorption at 280 nm was integrated based on a factor.

e ”280 (aprotinin) = 8.3

To obtain material for toxicological studies, the thrush strain KFN-1512 was grown on a large scale. The aprotinin analog was purified by combining reverse-phase ion exchange chromatography and HPLC.

Example II Production of (Glul, Glu42, Glu46) -aprotinin in the yeast strain KFN-1514.

From 10 oligonucleotides, a synthetic gene encoding (Glu 1, Glu42, Glu46) -aprotinin was synthesized by ligation, as described in Example I.

Plasmid pKFN-1505, obtained from plasmid pTZ19R, containing a synthetic gene linked in reading frame to a synthetic yeast leader peptide, was constructed as described in Example I.

Proceeding as in Example 1, the yeast expression plasmid pKFN-1510 was obtained, containing the following structure: TPIp - LaC212spx3 (1-47) -GluArgLeuGluLysArg (Glul, Glu42, Glu46) -aprotinin-TPIt signal-leader sequence.

The DNA sequence of the 412 bp EcoRI-XbaI fragment of plasmids pKFN-1505 and pKFN1510 is given under identification number 3 in the sequence list.

Plasmid pKFN-1510 was transformed, as described above, yeast strain MT663 to give yeast strain KFN-1514.

Cultivation of the transformed KFN-1514 strain in YPD medium, analysis of the amount of (Glul, Glu42, Glu46) -aprotinin in the supernatant and preparation of the material for toxicological studies was carried out as described above.

Example III. Production of (Glu42, Glu46) -aprotinin in the yeast strain KFN-1544.

The 144 bp AvaII-XbaI fragment encoding (Glu42, Glu46) -aprotinin (12-58) from pKFN-1505 was used to replace the corresponding DNA fragment encoding aprotinin (12-58) of the plasmid pKFN-1000, resulting in the plasmid pKFN-1000 1528. Plasmid pKFN-1 ° 0 ° is described in Example IV of International Patent Application, Publication No. WO 90/10075.

Proceeding as in Example 1, the yeast pKFN-154i expression plasmid was obtained, containing the following structure: TPIp - LaC212spx3 (1-47) -GluArgLeuGluLysArg (Glu42, Glu46) -aprotinin - TPIt signal-leader sequence.

The DNA sequence of the 412 bp EcoRI-XbaI fragment of plasmids pKFN-1528 and pKFN-1541 is given in the sequence list under the identification number.

Plasmid pKFN-1541 was transformed, as described above, yeast strain MT663 to give yeast strain KFN-1544.

Culturing the transformed KFN-1514 strain in YPD medium, analyzing the amount of (Glu42, Glu46) -aprotinin in the supernatant and preparing the material for toxicological studies was performed as described above.

Example IV Production (Ser10, Asp24, Thr26, Glu31, Asn41, Glu53) -aprotinin in the yeast strain KFN-1545.

A synthetic coding gene (Series, Asp24, Thr26, Glu31, Asn41, Glu53) -aprotinin was constructed from 10 oligonucleotides by ligation, as described in Example I.

Plasmid pKFN-1530, obtained from plasmid pTZ19R, containing a synthetic gene linked in reading frame to the synthetic yeast leader peptide sequence, was constructed as described in Example I.

Proceeding as in Example 1, the yeast expression plasmid pKFN-1532 was obtained, containing the following structure: TPIp - LaC212spx3 (1-47) -GluArgLeuGluLysArg signal leader sequence (Seri0, Asp24, Thr26, Głu31, Asn41, Glu53) -aprotinin - TPIt.

The DNA sequence of the 412 bp EcoRI-XbaI fragment of plasmids pKFN-1530 and pKFN1532 is given in the sequence list under identification number 7.

Plasmid pKFN-1532 was transformed as described above for the yeast strain MT663 to give the yeast strain KFN-1545.

168 250

Culturing the transformed KFN-1545 strain in YPD medium, analyzing the amount (Ser 10, Asp24, Thr26, Glu31, Asn41, Glu53) -aprotinin in the supernatant and preparing the material for toxicological studies was performed as described above.

P r ν V ł ** H Production plant T «miOO ΓΊΚ / ΛΠ Acrt / ll ClnAA Tvr4A \ _or> r / - \ 4-A7r» ir »* r W

X and £ - J 1 UM ». 1 IJV TIUl ^ kk.kkk ^ / XVJ XVVW4.V, »-_ · kj r \ ZJ 1 ΧΜΧΧ-Τ X,» n5 You XJ Χ ^ V »f Upl \ J \. J kkkkkj» »yeast strain KFN- 1547.

From 10 oligonucleotides, a synthetic gene encoding (Ser10, Leu20, Gly40, Asp41, Gln44, Tyr46) -aprotinin was ligated as described in Example 1.

Plasmid pKFN-1534, obtained from plasmid pTZ19R, containing a synthetic gene linked in reading frame to the synthetic yeast leader peptide sequence, was constructed as described in Example I.

Using the procedure of Example 1, the yeast expression plasmid pKFN-1537 was obtained, containing the following structure: TPIp - LaC212spx3 (1-47) -GluArgLeuGluLysArg signal (SerlO, Leu20, Gly40, Asn41, Gln44, Tyr46) - TProtin - TProtin.

The DNA sequence of the 412 bp EcoRI-XbaI fragment of plasmids pKFN-1530 and pKFN1537 is given in the sequence list under identification number 9.

Plasmid pKFN-1537 was transformed, as described above, yeast strain MT663 to give yeast strain KFN-1547.

Cultivation of the transformed KFN-1547 strain in YPD medium, quantity analysis (Ser10, Leu20, Glu40, Asn41, Gln44, Tyr46) -aprotinin in the supernatant and preparation of the material for toxicological studies was carried out as described above.

Example VI. Production of des-Argl, des-Pro2- (Ser42, Glu46) -aprotinin in the yeast strain KFN-1660.

Two fragments of the pKFN-306 plasmid: the 1.4 kb Ahall-Styl fragment and the 1.8 kb Ahall-Sall fragment were ligated into a duplex composed of the following two synthetic oligonucleotides:

NOR-2188: 5 'CAAGGCTGGTTTGTGTCAAACTTTCGTTTACGGTGGCTGCAGAGCTAAGTCCAACAACTTCGAATCTGCTGAAGACTGCATGAGAACTTGTGGTGGTGCCTAATCTAGAG 3'

NOR-2189: 5^J TCGACTCTAGATTAGGCACCACCACAAGTTCTCATGCAGTCTTCAGCAGATTCGAAGTTGTTGGACTTAGCTCTGCAGCCACCGTAAACGAAAGTTTGACACAAACCAGC 3 '

Plasmid pKFN-306 is a plasmid obtained from pTZ19R with a 502 bp EcoRI-XbaI insert, containing the signal peptide gene of the alpha and Saccharomyces cerevisiae conjugation factor linked in the reading frame to the synthetic de-Argl, des-Pro2- [Ser42] aprotinin gene . Construction of the plasmid pKFN-306 is described in WO 89/01968.

The ligation mixture was used to transform the competent E. coli (r ", m) strain. using ampicillin resistance selection. DNA sequencing (Sanger, F., Mieklen, S. and Coulsen, AR, Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467) showed that the plasmids from the resulting colonies contained the correct DNA sequence de-Argl, Pro2 - [Ser42, Glu46] -aprotinins.

One plasmid, pKFN-1629 was selected for further use.

Using the procedure of Example 1, the yeast expression plasmid pKFN-1656 was prepared containing the following construction: TPI_p - signal-leader sequence MFal (1-85) - dezArgl, dez-Pro2- [Ser42, Glu46] -aprotinin - TPIt16

168 250

The DNA sequence of the 502 bp EcoRI-XbaI fragment of plasmids pKFN-1629 and pKFN1656 is given in sequence II under identification number 11.

Plasmid pKFN-1656 was transformed, as described above, with the yeast strain MT663, multiplicity, m and uj (£ V μαμ ^ μζ, μ ™ j oLd-jo

Cultivation of the transformed KFN-1660 strain in YPD medium, analysis of the amount of des-Arg1, dJz-Pro2- (SJr42, Glu46) -aprotinin in the supernatant and preparation of the material for toxicological studies was performed as described above.

Example VII. Production of des-Arg1, des-Pro2- (Ser42, Ala46) -protinin in the yeast strain KFN-1661.

Two fragments of the pKFN-306 plasmid: the 1.4 kb AhaII-StyI fragment and the 1.8 kb AhaII-SalI fragment were ligated into a duplex composed of the following two synthetic oligonucleotides:

NOR-2196: 5 'CAAGGCTGGTTTGTGTCAAACTTTCGTTTACGGTGGCTGCA <GAGCTAAGTCCAACCAkCTTCGCTTCTGCTGAAGACTGCATGAGiAACTTGTGGTGGTGCCTAATCTAGAG 3'

NOR-2197: 5 'TCGACTCTAGATTAGGCACCACCACCAAGTTCTCATGCAGTCTTCAGCAGAAGCG2AAGTTGTTGGACTTAGCTCTGCAGCCACCGTAAACG2AAAGTTTGACACAAACCAGC 3'

Plasmid pKFN-306 is a plasmid obtained from pTZ19R with a 502 bp EcoRI-XbaI insert, containing the signal peptide gene of the alpha and Saccharomyces cerJvisiaJ signal conjugate fused in the reading frame to the synthetic de-Argl gene, des-Pro2 [Ser42j-aprotv. ins. Construction of the plasmid pKFN-306 is described in WO 89/01968.

The ligation mixture was used to transform the competent E. coli (r, m +) strain using ampicillin resistance selection. DNA sequencing (Sanger, F., Micklen, S. and Coulsen, AR, Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467) showed that the plasmids from the resulting colonies contained the correct DNA sequence de-Argl, dJz -Pto2- [SJt42, Glu46] -aprotinin.

One plasmid, pKFN-1631 was selected for further use.

Using the procedure of Example 1, the yeast expression plasmid pKFN-1657 was obtained, containing the following structure: TPIp - MFal (1-85) signal-leader sequence - dezArgl, dez-Pro2- [Ser42, Glu46] -aprotinin - TPIt.

The DNA sequence of the 502 bp EcoRI-XbaI fragment of plasmids pKFN-1631 and pKFN1657 is given under identification number 13 in the sequence list.

Plasmid pKFN-1657 was transformed, as described above, yeast strain MT663 to give yeast strain KFN-1661.

Cultivation of the transformed strain KFN-1661 in YPD medium, analysis of the amount of de-Argl, dJz-Pro2- (Ser42, Ala46) -aprotinin in the supernatant and production of material for toxicological studies was carried out as described above.

Example VIII. Production of des-Argl, des-Pro2- (Ser10, Asp24, Thrl6, Glu31, Asn41, Glu53) -aptotinin in the yeast strain KFN-1735.

A synthetic gene encoding de-Argl, dJz-Pro2- (Ser10, Asp24, Thr26, Glu31, Asn41, Glu53) -aprotinin was constructed from 10 oligonucleotides by ligation.

Plasmid pKFN-1707, obtained from plasmid pTZ19R, containing a synthetic gene linked in reading frame to a synthetic yeast leader peptide, was constructed as described in Example I.

Proceeding as in Example 1, the yeast expression plasmid pKFN-1709 was obtained, containing the following structure: TPIp - LaC212sp-3 (1-47) -GluArgLeuGluLysArg-dez-Argl, dJz-Pro2- (Ser10, Asp24, Thr26, signal-leader sequence) Glu31, Asn41, Glu53) aprotinin - TPIt.

168 250 17

The DNA sequence of the 406 bp EcoRI-XbaI fragment of plasmids pKFN-1707 and pKFN-1709 is given in the sequence list under identification number 15.

Plasmid pKFN-1709 was transformed as described above for the yeast strain MT663, ot_______: o_____j ______ ΤΧΤΓΑΤ n-łc

ULizyiii ^ cj> connection, cp uiuzuzuw) 1 ^ -1 incl.

The culture of transformed strain KFN-1735 in YPD medium, analysis of the amount of de-Argl, dez-Pro2- (Ser10, Asp24, Thr26, Glu31, Asn41, Glu53) -aprotinin in the supernatant and preparation of the material for toxicological studies was carried out as described above.

Example IX. Production of des-Arg 1, des-Pro2- (Asp24, Thr26, Glu31, Glu53) -aprotinin in the yeast strain KFN-1737.

Two pKFN-306 fragments: the 1.8 kb AhaII-XbaI fragment and the 1.4 kb Ahall-Avall fragment (see example V) were ligated to the 141 bp synthetic Avall-Xbal fragment encoding (Asp24, Thr26, Glu31, Glu53) -protimine.

The resulting plasmid, obtained from plasmid pTZ19R, was pKFN-1711.

Proceeding as in Example 1, the yeast expression plasmid pKFN-1713 was obtained, containing the following structure: TPIp - MF signal signal leader sequence al (1-85) - dezArgl, dez-Pro2- (Asp24, Thr26, Glu31, Glu53) -aprotinin - TPIt.

The DNA sequence of the 502 bp EcoRI-XbaI fragment of plasmids pKFN-1711 and pKFN-1713 is given in the sequence list under identification number 17.

Plasmid pKFN-1713 was transformed as described above, yeast strain MT663 to give the yeast strain KFN-1737.

Cultivation of the transformed strain KFN-1737 in YPD medium, analysis of the amount of de-Argl, de-Pro2- (Asp24, Thr26, Glu31, Glu53) -aprotinin in the supernatant and preparation of the material for toxicological studies was carried out as described above.

Example X. Production of des-Argl, dez-Pro2- (Ser10, Gly40, Asp41, Gly42, Glu53) aprotinin in yeast strain KFN-1739.

From 1 ° oligonucleotides by ligation, a synthetic gene encoding de-Argl, dez-Pro2- (Seri °, Gly40, Asn41, Gly42, Glu53) -aprotinin was constructed as described in Example I.

Plasmid pKFN-1751, obtained from plasmid pTZ 19R, containing a synthetic gene linked in reading frame to a synthetic yeast leader peptide, was constructed as described in Example I.

Proceeding as in Example 1, the yeast expression plasmid pKFN-1718 was obtained, containing the following structure: TPIp - LaC212spx3 (1-47) signal-leader sequence - GluArgLeuGluLysArg - dez-Argl, dez-Pro2- (Scri0, Leu20, Gly40, Asp41, Gly42, Glu53) aprotinin - TPIt.

The DNA sequence of the 406 bp EcoRI-XbaI fragment of plasmids pKFN-1715 and pKFN-1718 is given in the sequence list under identification number 19.

Plasmid pKFN-1718 was transformed as described above, yeast strain MT663, resulting in yeast strain KFN-1739.

The culture of transformed strain KFN-1739 in YPD medium, analysis of the amount of de-Argl, Pro2- (Seri °, Gly4 °, Asn41, Gly42, Glu53) -aprotinin in the supernatant and preparation of the material for toxicological studies was carried out as described above.

Example XI. Production of des-Argl, des-Pro2- (Ser42, Glu53) -aprotinin in yeast strain KFN-1742.

Two fragments of the pKFN-306 plasmid: AhaII-XbaI 1.8 kb long and 1.4 kb AhaII-AvaII fragment (see example V) were ligated with the 141 bp synthetic AvaII-XbaI fragment encoding (Ser42, Glu53) -aprotyninę.

The resulting plasmid obtained from plasmid pTZ19R was pKFN-1721.

Proceeding as in Example 1, the yeast expression plasmid pKFN-1724 was obtained, containing the following structure: TPIp - MFal (1-85) signal-leader sequence, de-Argl, de-Pro2- (Ser42, Glu53) -aprotinin - TPIt.

The DNA sequence of the 502 bp EcoRI-XbaI fragment of plasmids pKFN-1721 and pKFN-1724 is given in the sequence list under identification number 21.

Plasmid pKFN-1724 was transformed, as described above, yeast strain MT663 to give yeast strain KFN-1742.

168 250

Cultivation of the transformed KFN-1742 strain in YPD medium, analysis of the amount of de-Argl, dez-Pro2- (Ser42, Glu53) -aprotinmas in the supernatant and preparation of the material for toxicological studies was carried out as described above.

Example XII. Production of de-Argl, de-Pro2- (Glu42, Glu53) -protimic in yeast strain KFN-1752. '- - - Two fragments of the pKFN-306 plasmid: 1.8 kb AgaII-XbaI fragment and 1.4 kb AhaII-AvaII fragment (see example V) were ligated with a 141 bp synthetic AvaII-Xbal fragment encoding (Glu42, Glu53) -aptotyrlra.

The resulting plasmid obtained from plasmid pTZ19R was pKFN-1762.

Proceeding as in Example 1, the yeast expression plasmid pKFN-1765 was obtained, containing the following structure: TPIp - signal leader sequence MFα1 (1-85) - de-Argl, de-Pro2- (Ser42, Glu53) -aprotinin - TPIt.

The DNA sequence of the 502 bp EcoRI-XbaI fragment of plasmids pKFN-1762 and pKFN-1765 is given in the sequence list under identification number 23.

Plasmid pKFN-1765 was transformed as described above for the yeast strain MT663 to give the yeast strain KFN-1752.

Cultivation of the transformed KFN-1574 strain in YPD medium, analysis of the amount of de-Argl, de-Pro2- (Ser42, Glu53) -aprotinin in the supernatant and preparation of the material for toxicological studies was carried out as described above.

Example XIII. Production of des-Argl, des-Pro2- (Glu26, Ser42, Glu53) -aprotinin in yeast strain KFN-1755.

Two fragments of the pKFN-306 plasmid: AhaII-XbaI 1.8 kb long and 1.4 kb AhaII-AvaII fragment (see example V) were ligated with a synthetic 141 bp AvaII-XbaI fragment encoding (Glu26, Ser42, Giu53) -aprotymnę.

The resulting plasmid obtained from plasmid pTZ19R was pKFN-1768.

By proceeding as in Example 1, the yeast expression plasmid pKFN-1770 was obtained, containing the following structure: TPIp - MFα1 (1-85) signal-leader sequence - dezArgl, dez-Pro2- (Glu26, Ser42, Glu53) -aprotinma - TPIt.

The DNA sequence of the 502 bp EcoRI-XbaI fragment of plasmids pKFN-1768 with pKFN-1770 is given in the sequence list under identification number 25.

Plasmid pKFN-1770 was transformed, as described above, yeast strain MT663 to give yeast strain KFN-1755.

Cultivation of the transformed KFN-1755 strain in YPD medium, analysis of the amount of de-Argl, dez-Pro2- (Glu26, Ser42, Glu53) prototype in the supernatant and preparation of the material for toxicological studies was carried out as described above.

Example XIV. Production of de-Argl, de-Pro2- (Glu26, Glu42, Glu53) -aprotinin in yeast strain KFN-1756.

Two fragments of the pKFN-306 plasmid: the 1.8 kb AhaII-XbaI fragment and the 1.4 kb AhaII-AvaII fragment (see example V) were ligated with the 141 bp synthetic AvaII-Xbal fragment encoding (Glu26, Ser42, Glu53) -aprotyninę.

The resulting plasmid obtained from the plasmid pTZ19R was pKFN-1771.

Proceeding as in Example 1, the yeast expression plasmid pKFN-1773 was obtained, containing the following structure: TPIp - MF signal leader sequence α1 (1-85) - de-Argl, de-Pro2- (Glu26, Glu42, Glu53) -aprotinma - TPIt.

The DNA sequence of the 502 bp EcoRI-XbaI fragment of plasmids pKFN-1771 and pKFN-1773 is given in the sequence list under identification number 27.

Plasmid pKFN-1773 was transformed, as described above, yeast strain MT663 to give yeast strain KFN-1756.

Cultivation of the transformed KFN-1756 strain in YPD medium, analysis of the amount of de-Argl, dez-Pro2- (Glu26, Ser42, Glu53) -aprotinin in the supematant and preparation of the material for toxicological studies was carried out as described above.

Example XV Toxicological studies of aprotinin analogues when intravenously administered a single dose to Wistar rats.

Material.

168 250

The following analogues of aprotinin with reduced accidental load and thermal stability compared to recombinant aprotinin (1-58) were selected: KFN-1512, KFN-1514, KFN-1545, KFN-1547, KFN-1660, KFN-1661. Their main toxicological characteristics, characteristic features, are shown in Table 1. For comparison, data for recombinant aprotinin is provided. Temperature - denaturation was presented as an indicator of bilogical stability.

Table and General Information

<td>KFN type</td><td>Length chain</td><td>Cargo resultant</td><td>Temperature denaturation, ° C</td>

<td>rAprotynma</td><td> 1-58</td><td> + 6</td><td> &gt;100</td>

<td> 1512</td><td> 1-58</td><td> -2</td><td> 87</td>

<td> 1514</td><td> 1-58</td><td> 0</td><td> 88</td>

<td> 1544</td><td> 1-58</td><td> + 2</td><td> 98</td>

<td> 1545</td><td> 1-58</td><td> 0</td><td> 93</td>

<td> 1547</td><td> 1-58</td><td> + 2</td><td> 86</td>

<td> 1660</td><td> 3-58</td><td> + 2</td><td> 77</td>

<td> 1661</td><td> 3-58</td><td> + 3</td><td> 79</td>

<td> 1735</td><td> 3-58</td><td> -1</td><td> 68</td>

<td> 1737</td><td> 3-58</td><td> 0</td><td> 70</td>

<td> 1739</td><td> 3-58</td><td> + 1</td><td> 81</td>

<td> 1742</td><td> 3-58</td><td> + 2</td><td> 71</td>

<td> 1752</td><td> 3-58</td><td> + 1</td><td></td>

<td> 1755</td><td> 3-58</td><td> 0</td><td> 68</td>

<td> 1756</td><td> 3-58</td><td> -1</td><td> 70</td>

Test report.

On the first day of testing each analogue, a group of 2 male and 2 female rats received 33, 100,300 or 900 mg of analog / kg body weight. Two similarly formed control groups received saline or saline acidified with hydrochloric acid to a pH of about 4.5. This second solution served as a medium. The dose volume was 10 ml / kg body weight in all cases. Rats were observed for 7 days and killed on the eighth day. During the section, the kidneys were weighed and prepared for histopathology. Observed variables are presented in the header of Table 2.

Results.

The results of individual studies are summarized in Table 2. For comparison, data for recombinant aprotinin (dose: 11-300 mg / kg) were included, KFN-1512 could not be dissolved at the concentration required for the highest dose (900 mg / kg). One animal died at a dose of 900 mg KF'N-1545 / kg. In addition, no fatalities were observed.

No histopathological changes in the kidneys were observed after administration of KFN-1512, KFN-1544, KFN-1545 and KFN-1660 (300 mg / kg body weight). In addition, no histopathological changes in the kidneys were observed after administration of 900 mg / kg body weight KFN-1514, KFN-1547 and KFN-1661. Thus, the level of absence of toxic effects for all analogues was 300 mg / kg or more, compared with 11 mg / kg for aprotinin. Regarding the other variables observed, the analogs were equal to or exceeded aprotinin.

068 25°

Table 2

No toxic effects levels of observed variables, mg / kg

<td rowspan="3">KFN type</td><td colspan="8"></td>

<td colspan="3">J</td><td rowspan="2">Mortality</td><td rowspan="2">Observations macroscopic</td><td rowspan="2">Observations microscopic</td><td rowspan="2">Body weight Day 8</td><td rowspan="2">Kidney weight Day 8</td>

<td>0-30 min after the dose</td><td>2h after dose</td><td>daily</td>

<td>rApro-</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>tymna¹2</td><td> 33</td><td> 300</td><td> 300</td><td> 300</td><td> 33</td><td>AT</td><td> 100</td><td> 100</td>

<td> 1512’</td><td> 33</td><td> 300</td><td> 300</td><td> 300</td><td> 300</td><td> 300</td><td> 300</td><td> 300</td>

<td> 1514</td><td> 900</td><td> 900</td><td> 900</td><td> 900</td><td> 900</td><td> 900</td><td> 900</td><td> 900</td>

<td> 1544</td><td> 33</td><td> 100</td><td> 100</td><td> 900</td><td> 300</td><td> 300</td><td> 900</td><td> 300</td>

<td> 1545</td><td> 33</td><td> 900</td><td> 300</td><td> 300</td><td> 300</td><td> 300</td><td> 300</td><td> 300</td>

<td> 1547</td><td> 33</td><td> 300</td><td> 900</td><td> 900</td><td> 900</td><td> 900</td><td> 900</td><td> 900</td>

<td> 1660</td><td> 300</td><td> 900</td><td> 900</td><td> 900</td><td> 900</td><td> 900</td><td> 900</td><td> 900</td>

<td> 166’</td><td> 100</td><td> 900</td><td> 900</td><td> 900</td><td> 300</td><td> 900</td><td> 900</td><td> 900</td>

^{1 2} The highest dose = 300 mg / kg ² The smallest dose = 11 mg / kg

Conclusions.

The toxicity profile of the analogs, as estimated in studies of Wistar rats with a single administered dose, improved to a different extent compared to the toxicity profile of aprotinin. All aprotinin analogues had a nephrotoxic level of 300 mg / kg or more. The level without toxic effect for KFN-1514 was 900 mg / kg and was equal to the highest dose.

Example XVI. Elimination and distribution of recombinant aprotinin and aprotinin analogues.

Materials.

The recombinant authentic aprotinin and analogues prepared according to Examples I-VII were dissolved in 0.9% NaCl to obtain a dose volume of 1 µl / g rat weight. Injection solution concentrations were checked by analysis according to the methods given in the "Methods" section.

Methods.

Female Wistar rats weighing 200-300 g were used. Aprotinin and its analogs were tested using two different models: '/ anesthetized rats and 2 / meusypated rats.

Rats are anesthetized.

Rats anesthetized by intraperitoneal injection of sodium pentobarbital. The carotid artery and jugular vein were exposed and catheterized with polyethylene coils (PE-50, Intramedic). The carotid artery catheter was combined with a perfusor (B. Braun) for infusion with 3.8 ml 0.9% NaCl / h and with a blood pressure meter. Changes in blood pressure were recorded using a BD9 recorder (Kipp &amp; Zonen). The analogs were administered as an injection through a jugular vein catheter over 15 seconds.

- Blood samples were taken from the carotid artery catheter 3, 10, 20, 40 and 60 minutes after administration. Samples (0.45 ml) were collected in 3 ml samples containing 50 μΐ 0.13 M sodium citrate and centrifuged. The plasma was stored until analysis at -20 ° C. 60 minutes after administration, rats were killed with an excessive dose of sodium pentobarbital, and the kidneys and liver were removed, weighed and stored at -80 ° C.

Non-sleeping rats.

Before administration of the analogs, an oral dose of 2 ml distilled H2O was given. The analogs were administered intravenously as tail vein injections using an intravenous channel (Venflon 22 G, Viggo-Spectramet, Helsingborg, Sweden). After administration, the catheter was flushed with 0.5 ml 0.9%. NaCl 1 was removed. A patch was used to avoid tail bleeding at the injection site.

To collect the urine produced, the rat was then placed in a metabolic cage. After 3 hours, the rat was killed by CO 2 / O 2 (9/1) cage, and the kidneys and liver were removed and stored at -80 ° C until analysis. During CO 2 administration, the rat emptied bladder 1, after removing the animal, the cage was washed with 0.9% NaCl to obtain a total urine volume of 25 ml.

Preparation of homogenates.

One kidney (about 1 g) and about 2 g liver tissue were placed in separate 10 ml plastic tubes and 2 ml 0.9% NaCl was added. Tissues were homogenized 5 minutes using High

168 250

Intensity Ultrasonic Processor (Model VC50, Solics &amp; Materials Inc. Danbury CT, USA). Kidney and liver homogenates were diluted with saline to obtain a total volume of 10-25 and 4 ml, respectively.

A Λat i τ n η li τ-ττ r * XVLVZVi.j UUailLy.

Concentrations of aprotinin and analogues in plasma, liver homogenates and injection solutions were measured photometrically on Cobas Fara II (Roche). Briefly, plasma, homogenates and injection solutions were acid precipitated to remove non-aprotinin kallikrein inhibitors. Kallikrein inhibitory activity in the sample was measured using porcine pancreatic kallikrein (Sigma K 3627) and chromogenic substrate S2266 (Kabi). Kidney and urine homogenate concentrations were measured by the same method, except for the precipitation step, because the endogenous kallikrein inhibitory activity in diluted homogenates and urine was negligible. A separate standard curve was used for each analogue.

Test report.

14 groups of anesthetized rats and 14 groups of non-anesthetized rats were tested, each rat given a dose of i, 56pmol (about 10mg) aprotinin or aprotinin analogue per kg body weight. Basic data on 28 groups are given in Table 3.

Table 3

<td>group</td><td>n</td><td>Toilets</td><td>WN</td><td>WW</td>

<td>rAprotymna U</td><td> 5</td><td> 251,8</td><td> 0,98</td><td> 9,9</td>

<td>KFN 1512 U</td><td> 4</td><td> 230,0</td><td> 0,84</td><td> 9,6</td>

<td>KFN 1514 U</td><td> 4</td><td> 220,5</td><td> 0,85</td><td> 8,6</td>

<td>KFN 1544 U</td><td> 4</td><td> 226,0</td><td> 0,97</td><td> 9,3</td>

<td>KFN 1545 U</td><td> 4</td><td> 224,8</td><td> 0,92</td><td> 9,2</td>

<td>KFN 1547 U</td><td> 4</td><td> 229,0</td><td> 0,75</td><td> 8,6</td>

<td>KFN 1660 U</td><td> 4</td><td> 220,5</td><td> 0,93</td><td> 9,7</td>

<td>KFN 1661 U</td><td> 4</td><td> 240,8</td><td> 0,97</td><td> 9,0</td>

<td>rAprotymna N</td><td> 4</td><td> 192,5</td><td> 0,77</td><td> 9,8</td>

<td>KFN 1512 N.</td><td> 5</td><td> 191,0</td><td> 0,65</td><td> 7,2</td>

<td>KFN 1514 N.</td><td> 6</td><td> 188,3</td><td> 0,69</td><td> 7,3</td>

<td>KFN 1544 N.</td><td> 5</td><td> 190,0</td><td> 0,64</td><td> 6,5</td>

<td>KFN 1545 N.</td><td> 6</td><td> 185,8</td><td> 0,66</td><td> 7,3</td>

<td>KFN 1547 N.</td><td> 5</td><td> 188,0</td><td> 0,67</td><td> 7,3</td>

<td>KFN 1660 N.</td><td> 6</td><td> 205,0</td><td> 0,77</td><td> 8,5</td>

<td>KFN 1661 N.</td><td> 6</td><td> 204,2</td><td> 0,80</td><td> 8,1</td>

WC body weight (g) WN kidney weight (g)

WW liver weight (g) U model of anesthetized rats N model of anesthetized rats

Statistical analysis.

For statistical analysis, the Spearman sum order correlation test was used.

Table 4

Aprotinin analogues content of inhibitory activity in the kidneys and urine after intravenous administration to rats

<td>Analog</td><td>Cargo resultant</td><td>Temperature denaturation²° C (max)</td><td>Urine content (3 h) % of dose</td><td>Kidney content (lh) % of dose</td><td>Kidney content (3h) % of dose</td><td>Indicator accumulation "</td>

<td>aprotinin</td><td> + 6</td><td> &gt;100</td><td> 2</td><td> 21</td><td> 44</td><td> 2,10</td>

<td>KFN 1512 "</td><td> -2</td><td> 87</td><td> 51</td><td> 2</td><td> 2</td><td> 1</td>

<td>KFN 1514</td><td> 0</td><td> 88</td><td> 36</td><td> 8</td><td> 11</td><td> 1,38</td>

<td>KFN 1544</td><td> + 2</td><td> 98</td><td> 42</td><td> 17</td><td> 23</td><td> 1,35</td>

<td>KFN 1545</td><td> 0</td><td> 93</td><td>4i</td><td> 14</td><td> 28</td><td> 2</td>

<td>KFN 1547</td><td> + 2</td><td> 86</td><td> 45</td><td> 4</td><td> 5</td><td> 1,25</td>

<td>KFN 1660</td><td> + 2</td><td> 77</td><td> 23</td><td> 6</td><td> 3</td><td> 0,5</td>

<td>KFN 1661</td><td> + 3</td><td> 79</td><td> 18</td><td> 4</td><td> 3</td><td> 0,75</td>

The rate of renal accumulation was calculated as the content of inhibitory activity after 3 hours divided by the content after and hour. Measured by differential scanning calorimeter in 20 mM 2- (N-morphdlino) ethanesulfonic acid

168 250

Results.

Analogues in the kidneys and urine.

The total content in the kidneys (in percentage of the dose) after 1 and 3 hours and in the urine after 3 families is shown in Fig. 3 and Table 4. It appears that large τóznlcr was found between the analogues.

Regarding aprotinin, the content in the kidneys 1 hour after administration was approximately 20% of the dose, while the content increased to more than 40% after 3 hours. Disadvantage ^ no apTotynmy in urine was insignificant.

In order to assess whether urinary excretion after 3 hours was associated with the analogue accident load, a degree of correlation between these values ​​was calculated.

It was found that the content in urine was strongly correlated with the net charge of analogues (see Fig. 4).

Table 5

<td>analogs</td><td>Accumulation indicator</td><td>Stability index in the kidneys</td><td>Denaturation temperature (° C)</td>

<td>rAprotinin A</td><td> 2,10</td><td> 0,90</td><td> 100</td>

<td>KFN 1512 A</td><td> 0,66</td><td> 0,67</td><td> 87</td>

<td>KFN 1514 A</td><td> 1,32</td><td> 0,87</td><td> 88</td>

<td>KFN 1544 A.</td><td> 1,32</td><td> 1,05</td><td> 98</td>

<td>KFN 1545 A</td><td> 1,99</td><td> 0,71</td><td> 93</td>

<td>KFN 1547 A</td><td> 1,22</td><td> 0,65</td><td> 86</td>

<td>KFN 1660 A</td><td> 0,50</td><td> 0,55</td><td> 77</td>

<td>KFN 1661 A</td><td> 0,77</td><td> 0,55</td><td> 79</td>

Analogue stability.

To study the stability of analogues in kidney tissue, one kidney from 14 anesthetized rats (one from each group) was divided into two parts with identical weights. One part was stored at 37 ° C and the other at 4 ° C. After four hours, the tissues were homogenized and the content of analogs measured. The stability index was defined as the contents in the part stored at 37 ° C divided by the contents in the part stored at 4 ° C. Stability indices are given in Table 6. They show that TApTotinin, KFN 1514 and KFN 1544 appear to be the most stable compounds, compared to, for example, KFN 1660, which turned out to be more unstable.

The stability of analogues was also studied by determining their denatation temperature. Denaturation temperatures were found to be well correlated with content in the kidney tissue three hours after administration (Fig. 5) and with the accumulation index (Fig. 6), but not with urinary excretion. These data suggest that the net charge may be important for urinary excretion, but less important for renal concentration and accumulation. On the other hand, accumulation in the kidneys appears to be related to the denaturation temperature and stability of the analogues in kidney tissue. However, it is likely that concentrations measured in kidney tissue 1 hour after administration have changed due to degradation or movement. Thus, it is possible that concentrations measured e.g. 10 minutes after administration will correlate with the accident load.

Conclusions. The following conclusions were drawn:

1) All tested analogs were taken by the kidneys, but to varying degrees. Accumulation in the kidneys appears to be related to thermal stability and stability in the kidney tissue, but not to the net charge of the molecule.

2) Urinary excretion seemed to be related to the analogue accident load, but not to stability.

168 250

SEQUENCE LIST (1) GENERAL INFORMATION:

(and)

NOTIFYING: Bjoern, Soeren Erik Noms, Kjeld Diness, Viggo Noerskov-Lauritsen, Leif Cłwistensen, Niels Dyhr Bregengaard, Claus (ll) TITLE OF THE INVENTION: Aprotinin analogues (lii) NUMBER OF SEQUENCES. 29 (1V) ABRES TO:

(A) ADDRESS: Novo Nordisk A / S (B) STREET: Novo Alle (C) LOCATION: Bagsvaerd (D) COUNTRY: Denmark (E) POST CODE: 2880 (v) COMPUTER ENTRY (A) MEDIA TYPE: Diskette (B) ) COMPUTER: IBM and IBM (C) OS: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Release # 1.0, version 1. 25 (vi) CURRENT DATA APPLICATIONS:

(A) APPLICATION NUMBER:

(B) SUBMISSION DATE:

(C) CLASSIFICATION:

(vii) DETAILS OF A PREVIOUS NOTIFICATION:

(A) DECLARATION NUMBER: DK 2361/90 (B) SUBMISSION DATE: October 1, 1990 (vii) DETAILS OF A PRIOR ENTRY:

(A) DECLARATION NUMBER: DK 1118/91 (B) DATE OF SUBMISSION: June 12, 1991 (vill) INFORMATION ON THE FULL MOTOR / AGENCY (A) NAME: Tłialsoe-Madsen, Bngit (C) REFERENCE / REUESTER NUMBER: 3465. 204-- W0 (ix) TELECOMMUNICATIONS INFORMATION: (A) TELEPHONE: (212) 867-0123 (B) TELEFAX: (212) 0857-0298 (C) TELEX

(2) INFORMATION ABOUT SEQUENCE NUMBER 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 418 base pairs (B) TYPE: nucleic acid (C) NUMBER OF THREADS: one (D) TOPOLOGY: linear

168 250

<td> (11)</td><td>TYPE</td><td>PARTICLES: cDNA</td>

<td>(Vi)</td><td>SOURCE (A \ (Al</td><td>ORIGINAL:</td>

<td>(1X)</td><td>FEATURE: (AND) (B)</td><td>NCZWA / KLUTZ: CDS LOCATION: 77..409</td>

<td>(1X)</td><td>FEATURE: (AND) (B)</td><td>NAME / KEY. syg_peptyd. LOCATION: 77..235</td>

<td>(1X)</td><td>FEATURE: (AND) (B)</td><td>NAME / KEY: mat peptide LOCATION: 236..409</td>

<td>(X1)</td><td colspan="2">DESCRIPTION OF THE SEQUENCE: SEQ ID No. 1:</td>

GAATTCCATT TACGACTAGT TTCACCCMC CGMTCTCAA TCMTCACT CATCCCCCM 60

AIAAAOGACC ACCAGC CCG AAG GCT GCT CCC TTG GIT TTG TCC TTG CCT 109

Mat Lys Ma Val Phe Leu Val leu Cheese Leu Ile

-53 -50 -45

<td colspan="3">GGA TTC CGT</td><td colspan="2">TTG GCC CMC</td><td rowspan="2">CCC Pro</td><td colspan="4">GTC CCC GGC GCT GAA CCA TCC GIC GAG</td><td rowspan="2"> 1^57</td>

<td>Gly</td><td>phe</td><td>Cys -40</td><td>Trp Ala</td><td>dn</td><td>Val Thr -35</td><td>dy Asp</td><td>du</td><td>Cheese Cheese Val du -30</td>

<td>ATT</td><td>COG</td><td>DCC</td><td>(GA TT</td><td>CCTG</td><td>ATC</td><td>ATC GT</td><td>&lt;GA MC</td><td>ACCC</td><td>ACT TC! GTT CCC</td><td> 205</td>

<td>How much</td><td>Pro -25</td><td>Gilu</td><td>Gilu Cheese</td><td>Hau</td><td>How much -twenty</td><td>How much has he got</td><td>Gilu Asn</td><td>cr -15</td><td>Bff Leu ALa Asn</td><td></td>

<td>GTT</td><td>GCC</td><td>ACG</td><td>(GCT dC</td><td>ad</td><td>TT!</td><td>OA MA</td><td>GCC ad</td><td>(TTT</td><td>dAT TCC TTT TCT</td><td> 255</td>

<td>val -10</td><td>ALa</td><td>Underworld</td><td>Has a du</td><td>mg -5</td><td>How many</td><td>for Lys</td><td>Mg for 1</td><td>Pro</td><td>Mp Hie (Cys Leu 5</td><td></td>

<td>GAA</td><td>TTT</td><td>CTTC</td><td>TAC ACT</td><td>dT</td><td>CCCA</td><td>ICC? AA</td><td>dT Ad</td><td>ACTC</td><td>ACTC Ad TAC TCC</td><td> 330</td>

<td>Glu</td><td>Pro</td><td></td><td>(You Kur 10</td><td>dy</td><td>fto</td><td>&lt;Ts Lys 15</td><td>Ma Mg</td><td>How much</td><td>Ile Mg Τ / τ Rh twenty</td><td></td>

<td>TAC</td><td>AAC</td><td>GGT</td><td>GGA GGT</td><td>dT</td><td>TCG</td><td>TT CCA</td><td>act tc</td><td>GGIT</td><td>TAC GU dG TT</td><td> 334</td>

<td>Tyr</td><td>own</td><td>checkmate 25</td><td>du Ma</td><td>dy</td><td>Leu</td><td>cn dn 30</td><td>TCr IPe</td><td>val</td><td>ITs dy dy CTs 35 </td><td></td>

<td>AGA</td><td>GCT</td><td>GGA</td><td>AAA ACC</td><td>aa</td><td>TTC</td><td>GGA ITT</td><td>GCT GGA</td><td>GGC</td><td>TCT AAC Ad ACTT</td><td> 33</td>

<td>Arg</td><td>ala</td><td>du</td><td>Mg Mn</td><td>acs</td><td>phe</td><td>du See</td><td>Has a du</td><td>acs</td><td>Cts MML Mg LCh</td><td></td>

45 55

TGC GGT GGT GCC TACTTEAGC Cys Gly Gly Ala

168 250 (2) INFORMATION ABOUT SEQ ID No. 2:

(l) SEQUENCE CHARACTERISTICS:

<td></td><td>(AND)</td><td>νιυϋυον: ni amino * waso in</td>

<td></td><td>(B)</td><td>TYPE · amino acid</td>

<td></td><td>(D)</td><td>TOPOLOGY: linear</td>

<td> (11)</td><td>TYPE</td><td>PARTICLES: Diałko</td>

<td>(Xi)</td><td colspan="2">SEQUENCE DESCRIPTION: NUMBER SEQUENCE</td>

IDENTIFICATION 2:

Met Lys Ala Val Ehe Leu Val Leu Cheese Leu Ile Gly Ehe Cys Trp Ala

-53 -50 -45 -40

Gln Pro Val Ihr Gly Asp Glu Cheese Cheese Val Glu Ile Pro Glu Glu Cheese

-35 -30 -25

Leu Ile Ile Ala Glu Asn Thr Thr leu Ala Asn Val Ala Met Ala Glu -20 -15 -10

Arg leu Glu Lys Arg Glu Pro Asp Phe Cys leu Glu Pro Pro Tyr Ihr

-5 1 5 10

Gly Pro Cys Lys Ala Arg Ile Ile Arg Tyr Phe Tyr Asn Ala Glu Ala

20 25

Gly Leu Cys Gln Thr Ehe Val Tyr Gly Gly Cys Arg Ala Glu Arg Asn 30 35 40

Asn Phe Glu Cheese Ala Glu Asp Cys Met Arg Ihr Cys Gly Gly Ala 45 50 55 (2) SEQUENCE INFORMATION

ABOUT IDENTIFICATION NUMBER 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 418 base pairs (B) TYPE. Nucleic acid (C) NUMBER OF THREADS: one (D) TOPOLOGY: linear

<td>(Ii)</td><td>TYPE</td><td>PARTICLES: cDNA</td>

<td>(VI)</td><td>SOURCE (AND)</td><td>ORIGINAL: ORGANISM: synthetic</td>

<td>(IX)</td><td>C c, CHA: (AND) (B)</td><td>NAME / KEY: CDS LOCATION: 7 7., 4 O9</td>

<td>(IX)</td><td>bC HA: tA) (B)</td><td>NAME / KEY. syg_oeptya LOCATION: 77 .235</td>

168 250 (ix) FEATURE:

(A) NAME / KEY: mat_pepTyd (B) LOCATION, 236,, 409 (X1) SEQUENCE DESCRIPTION: IDENTIFICATION NUMBER 3:

GAAnTCCAT CAAGAATAGT TCAAACAAGA AGUTACAAA CTAITAGTTT CATACACAAT 60

AIAAACGACC GGAGGA ATC AAG GTT GTT TTC TTT GIT TTT TCC TTT ATC 109

Mat Lys Ala Val Phe Leu Val Leu Cheese Leu Ile

-53 -50 -45

<td rowspan="2">GGA TTC Gly Phe</td><td rowspan="2">TCC Cys -40</td><td colspan="4">TGG (GCC CCA CCA GTC ATT GGC</td><td colspan="2">dG GZA TTT</td><td rowspan="2">TTC GGT dA See Vaa du</td><td rowspan="2"> 157</td>

<td>TTp Aa</td><td>dro Rro</td><td>val -35</td><td>Thr gly</td><td>af)</td><td>du Ser -30</td>

<td>ATT CCG</td><td>GAA</td><td>dG TTC?</td><td>CCT ATT</td><td>ATC</td><td colspan="2">ATT GAG ZAA</td><td>AAC AAT</td><td>TTC dT ZAA</td><td> 205</td>

<td>How much Pro</td><td>Glu</td><td>Gilu Cheese</td><td>Leu Ile</td><td>How much</td><td>ALa GLu</td><td>an</td><td>TTR</td><td>How many Aa Ζαπ</td><td></td>

<td> -25</td><td></td><td></td><td> -20</td><td></td><td></td><td></td><td> -15</td><td></td><td></td>

<td>GTC GTC</td><td>ATG</td><td>GCT dG</td><td>Ad TTC</td><td>cord</td><td>GAG AGA</td><td>GGA</td><td>GGA CCT</td><td>TTT TTT TTT</td><td> 253</td>

<td>Val Ala</td><td>checkmate</td><td>Αεί du</td><td>Ag Au</td><td>Gilu</td><td>Lys Arg</td><td>du</td><td>Pro Aas</td><td>EPh Ccs iLe</td><td></td>

<td> -10</td><td></td><td></td><td> -5</td><td></td><td></td><td> 1</td><td></td><td> 5</td><td></td>

<td>GAA CCT</td><td>CCA</td><td>tta AAT</td><td>GGT CTTA</td><td>TTTf</td><td>AGA ATT</td><td>AAG</td><td>AAT AAT</td><td>AAG TTA ITT</td><td> 301</td>

<td>du pro</td><td>Pro</td><td>You thr</td><td>dy EPo</td><td>CTs</td><td>Lys ALa</td><td>Aaj</td><td>How much</td><td>Aig Tts PPh</td><td></td>

<td></td><td></td><td> 10</td><td></td><td></td><td> 15</td><td></td><td></td><td> 20</td><td></td>

<td>TAC AAC</td><td>GCC</td><td>AGA GU¹</td><td>GGC -TT</td><td>TTT</td><td>CAA ATT</td><td>TTC</td><td>CTT TTA</td><td>GCT GGG TTT</td><td> 334)</td>

<td>Tyr Asn</td><td>ala</td><td>Lls Al</td><td>dy ILU</td><td>CTs</td><td>Gin Thr</td><td>PPh</td><td colspan="2">VVL Tyy dy dy Cys</td><td></td>

<td></td><td> 25</td><td></td><td></td><td> 30</td><td></td><td></td><td> 35</td><td></td><td></td>

<td>AGA GCT</td><td>AAG</td><td>GAA. AGA</td><td>AAA -TT</td><td>GGA</td><td>TTT GTT</td><td>dA</td><td colspan="2">dG TT AAT Ad AAT</td><td> 337</td>

<td>Arg ALa</td><td>lys</td><td>du aas</td><td>Aas ETt</td><td>du</td><td>Aa cheese</td><td>du</td><td colspan="2">Aap Cys Met Aaj TTr</td><td></td>

<td> 40</td><td></td><td></td><td> 44</td><td></td><td></td><td></td><td> 50</td><td></td><td></td>

<td>TGT GCT</td><td>GCT</td><td colspan="2">GCC TAGTClAd</td><td></td><td></td><td></td><td></td><td></td><td> 418</td>

Cys Gly Gly Ala 55 (2) SEQUENCE IDENTIFICATION NUMBER 4:

(I) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 111 am ^ noo — aso— (B) TYPE: amino acid (D) TOPOLOGY: linear (II) PARTICLE TYPE: protein (X1) SEQUENCE DESCRIPTION: IDENTIFICATION MUMMER SEQUENCE 4:

Me Lys Ala Val Phe Leu Val Leu Cheese Leu Ile Gly Phe CCs Aa

-53 -50 -45 -₄₀

168 250

<td>Gin</td><td>Pro</td><td>val</td><td>tm</td><td>dy</td><td>Asp</td><td>du</td><td>Cheese</td><td>Sar</td><td>val</td><td>du</td><td>How much</td><td>Pro</td><td>du</td><td>du</td><td>Cheese</td>

<td></td><td></td><td> -35</td><td></td><td></td><td></td><td></td><td> -30</td><td></td><td></td><td></td><td></td><td> -25</td><td></td><td></td><td></td>

<td>Leu</td><td>How much</td><td>Loam_e</td><td>ala</td><td>du</td><td>.Asn</td><td>Thr</td><td>Thi</td><td>Leu</td><td>Al a</td><td>own</td><td>val</td><td>AI_andthulium</td><td>Underworld</td><td>C Ti rtiŁi</td><td>du Glu</td>

<td></td><td> -20</td><td></td><td></td><td></td><td></td><td> -15</td><td></td><td></td><td></td><td></td><td> -10</td><td></td><td></td><td></td><td></td>

<td>Arg</td><td>Leu</td><td>Glu</td><td>lys</td><td>Arg</td><td>Glu</td><td>Pro</td><td>aro</td><td>phe</td><td>Cys;</td><td>How many</td><td>Glu</td><td>Pro</td><td>Pro</td><td>You</td><td>Thr</td>

<td> -5</td><td></td><td></td><td></td><td></td><td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td>

<td>Gly</td><td>Pro</td><td>Cys</td><td>lys</td><td>ala</td><td>aig</td><td>How much</td><td>How much</td><td>AAJ</td><td>You</td><td>PHH</td><td>You</td><td>Asa</td><td>ala</td><td>Forest</td><td>Ca</td>

<td></td><td></td><td></td><td> 15</td><td></td><td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td>

<td>dy</td><td>Leu</td><td>Cys</td><td>Gin</td><td>Thr</td><td>phe</td><td>val</td><td>You</td><td>dy</td><td>dy</td><td>Cys</td><td>Arg</td><td>ala</td><td>lys</td><td>du</td><td>own</td>

<td></td><td></td><td> 30</td><td></td><td></td><td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td>

<td>own</td><td>phe</td><td>du</td><td>Cheese</td><td>ala</td><td>du</td><td>Asp</td><td>Cys</td><td>Underworld</td><td>Arg</td><td>You</td><td>Cys</td><td>dy</td><td>dy</td><td>Ca</td><td></td>

<td></td><td> 45</td><td></td><td></td><td></td><td></td><td> 55)</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td>

(2) INFORMATION ON SEQUENCE IDENTIFICATION NUMBER 5:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 418 base pairs.

(B) TYPE: nucleic acid (C) NUMBER OF THREADS: one (D) TOPOLOGY: linear (11) PARTICLE TYPE: CDNA

<td>(V1)</td><td>SOURCE (AND)</td><td colspan="2">ORIGINAL: ORGANISM: synthetic</td>

<td>(1X)</td><td>FEATURE: (AND)</td><td>NTZWA / KEY:</td><td>CDS</td>

<td></td><td>(B)</td><td>LOCATION:</td><td> 77..409</td>

<td>(1X)</td><td>FEATURE: (AND)</td><td>NTZWA / KEY:</td><td>syg peptide</td>

<td></td><td>(B)</td><td>LOCATION:</td><td> 77..235</td>

<td>(1X)</td><td>FEATURE: (AND)</td><td>NTZWA / KEY:</td><td>mat_peptyd</td>

<td></td><td>(B)</td><td>LOCATION:</td><td> 236..409</td>

(X1) DESCRIPTION OF THE SEQUENCE: SEQUENCE WITH THE IDENTIFICATION NUMBER 5;

GAATTCCATT CAAGAATAGT TTCAA ^ CCA ^ GG. AGGALYCCAA. CCTACCATTT CTATCCCCAC

ATCCAdCCC CTACGC ATG ATT GGT GGT TTC ITT GGT ΊΊΓ TCC TTC ACT

Met Lyy Ma Val PPe Leu Val leu Cheese Llu Ile

-53 -50 -45

GGC TTC TC TC &lt;CC CTA CTA CGT ACC CCC CGA GGT TCC TT (GIT GAG

Gly Phe Q / s Τη? Ma Gin IPo VVL TTr Gil AAp Glu S &amp; e Ser W Glu

-40 -35 -30

109'

157

168 250

<td colspan="9">ATT CCG GAG &lt;GG TTG? CTG ATC ATC GG? GM MC ATC AIG TTC TCT MG</td><td rowspan="2"> 22^&lt;5</td>

<td>How much</td><td>Pro Glu Gilu Cheese -25</td><td>Lu Ile -20</td><td>How much has he got</td><td>Glu</td><td>Mn TCh T? R -15</td><td>Lu</td><td>has</td><td>tone</td>

<td>GTC</td><td>GCC ATC CGG GM</td><td>AG TTC</td><td>cm MG</td><td>ACH</td><td>ACH '(GG? GGA</td><td>TTTG</td><td>TTG?</td><td>TTG</td><td> 253</td>

<td>val</td><td>Ala Met Ma Glu</td><td>Leu tog</td><td colspan="2">Glu Lys tog</td><td>7ATJ M top</td><td>Rie</td><td>CCS</td><td>leu</td><td></td>

<td> -10</td><td></td><td> -5</td><td></td><td></td><td> 1</td><td></td><td> 5</td><td></td><td></td>

<td>GAA</td><td>TCT GGG TTCG AAT</td><td>(Gd? CCA</td><td>TTG? MA</td><td>GG?</td><td>ACH AAC MT</td><td>AIG.</td><td>TTG</td><td>TTC</td><td> 301</td>

<td>how many</td><td>Pro Pro iyy TCh</td><td>Gly Pro</td><td>CG-'S Lys</td><td>has</td><td>Mg Ile Ile</td><td>.track</td><td>TTS</td><td>fhe</td><td></td>

<td></td><td> 10</td><td></td><td> 15</td><td></td><td></td><td> 20</td><td></td><td></td><td></td>

<td>TAC</td><td>AAC GCC MG GlG</td><td>απ 'ttg</td><td>TTG? CCA</td><td>AAG?</td><td>TTG GGT TTG</td><td>GGG</td><td>GGG</td><td>TTG</td><td> 3319</td>

<td>Tyr</td><td>Asn ALa Lys Ma</td><td>Gly Leu</td><td>CCS Gin</td><td>T? R</td><td colspan="3">Rie Val Tts Gly dy</td><td>cgs</td><td></td>

<td></td><td> 25</td><td></td><td> 30</td><td></td><td> 35</td><td></td><td></td><td></td><td></td>

<td>AGA</td><td>GCC GGl GM MA</td><td>MC TTG</td><td>dA TTG?</td><td>&lt;dT</td><td colspan="2">CGA GGA TTG AAT</td><td>* HAG</td><td>* / —Tn HAG</td><td>o - t 03 9</td>

<td>Arg</td><td>Ma Lys Glu Mn</td><td>Phe ton</td><td>du cheese</td><td>has</td><td colspan="3">du Aa? CCS Met Aa.</td><td>ITH</td><td></td>

<td></td><td> 40</td><td> 45</td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td>

<td>TCT</td><td colspan="2">GGT GGT GCC TAGTCHiG</td><td></td><td></td><td></td><td></td><td></td><td></td><td> 418</td>

Cys Gly Gly Ala 55 (2) SEQUENCE ID No. 6:

(I) SEQUENCE CHARACTERISTICS:

(A) LENGTH: m amino acids (B) TYPE: amino acid (D) TOPOLOGY: Contract (II) PARTICLE TYPE: protein (X1) SEQUENCE DESCRIPTION: SEQUENCE

<td></td><td></td><td></td><td>IDE</td><td>NTY '</td><td colspan="2">FIKGGYJ</td><td>'NYM</td><td> 5:</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Mayor</td><td rowspan="2">lys</td><td>has</td><td>val</td><td>PPh</td><td>leu</td><td>val</td><td>irsl</td><td>sst</td><td>Lsu</td><td>lle</td><td>Gly</td><td>phe</td><td>lys</td><td>ehe</td><td>sr?</td>

<td> -53</td><td></td><td> -55</td><td></td><td></td><td></td><td></td><td> -45</td><td></td><td></td><td></td><td></td><td> -40</td><td></td><td></td>

<td>dn</td><td>Pro</td><td>IN</td><td>htt</td><td>Gly</td><td>Asp</td><td>gpg</td><td>UES</td><td>sst</td><td>val</td><td>du</td><td>iue</td><td>Pro</td><td>Glu</td><td>Glu</td><td>Sei</td>

<td></td><td></td><td> -35</td><td></td><td></td><td></td><td></td><td> -30</td><td></td><td></td><td></td><td></td><td> -25</td><td></td><td></td><td></td>

<td>Lsu</td><td>How much</td><td></td><td>has</td><td>du</td><td>own</td><td>The</td><td>hcr</td><td>leu</td><td>has</td><td>own</td><td>VAA</td><td>has</td><td>Ael</td><td>To me</td><td>Glu</td>

<td></td><td> -20</td><td></td><td></td><td></td><td></td><td> -15</td><td></td><td></td><td></td><td></td><td> -10</td><td></td><td></td><td></td><td></td>

<td>Arg</td><td>leu</td><td>du</td><td>LLS</td><td>Arg</td><td>mg</td><td>Pro</td><td>Asp</td><td>phe</td><td>CCS</td><td>Leu</td><td>Glu</td><td>Pro</td><td>Pro</td><td>TCs ·</td><td>uh</td>

<td> — 5</td><td></td><td></td><td></td><td></td><td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td>

<td rowspan="2">Gly</td><td>Pro</td><td>cvs</td><td>Lyy</td><td>has</td><td>Arg</td><td></td><td>How much</td><td>Arg</td><td>ITyr</td><td>lhe</td><td>Tyr</td><td>own</td><td>ALa</td><td>lys</td><td>ml</td>

<td></td><td></td><td> 15</td><td></td><td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td>

<td>dy</td><td>Leu</td><td>Cys;</td><td>Gin</td><td>TTR</td><td>phe</td><td>VAV</td><td>T ^ c</td><td>Gly</td><td>Gly</td><td>Gys</td><td>srg</td><td>has</td><td>.Lys</td><td>G1l</td><td>Ag</td>

<td></td><td></td><td> 30</td><td></td><td></td><td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 4(0</td><td></td><td></td><td></td>

<td>own</td><td>fhe</td><td>du</td><td>See</td><td>has</td><td>Glu</td><td>ma)</td><td>CCS</td><td>Underworld</td><td>mg</td><td>Thr</td><td>(Quoted</td><td>Gly</td><td>Gly</td><td>has</td><td></td>

<td></td><td> 45</td><td></td><td></td><td></td><td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td>

068 25 ° 29 (2) INFORMATION ABOUT SEQUENCE ID No. 7:

(1) SEQUENCE CHARACTERISTICS:

<td rowspan="2"></td><td rowspan="2">(AND) (B) (C) (D)</td><td colspan="2">LENGTH: 418 base pairs. AYP: Acid nuc ^ e. ^ And ^ (^ ex</td>

<td>NUMBER OF THREADS: TOPOLOGY: L.</td><td>one iniowa</td>

<td> (11)</td><td>TYPE</td><td colspan="2">PARTICLES: cDNA</td>

<td>(V1)</td><td>ZRCDŁO (AND)</td><td colspan="2">ORIGINAL: ORGANISM: synthetic</td>

<td>(1X)</td><td>FEATURE: (AND) (B)</td><td>NAME / KEY: LOCATION:</td><td>CDS 77..409</td>

<td>(1X)</td><td>FEATURE: (AND) (B)</td><td>NAME / KEY: LOCATION,</td><td>syg_peptide; 77..500</td>

<td>(1X)</td><td>FEATURE: (AND) (B)</td><td>NAME / KEY: LOCATION:</td><td>mat_peptyd 236..409</td>

(X1) DESCRIPTION OF THE SEQUENCE: SEQUENCE WITH THE IDENTIFICATION NUMBER 7:

GAATTCGATT CAAGAATAGT TCAAACAATA ACAITACAAT CTATCAATTT CATACACAAT 60

AIAAACGACC AAAACA AAT AAC CCA GIT TAC TTG GIT TAC TCC TIT AAC 109

Met Lys ALa Val Ehe Leu VaL Leu Cheese Leu Ile

-53 -50 -45

GGA TTC TCC TGS GC CAA ACA CCC ACT GGT CC AC TCTc ACTGIT TAG 157

GLy Hie Cys Trp ALa Gin Pro V &lt;= V Ua Gic Asa GIt uer Ser Val Gic

-40 335 -30

ATT CCC CCA GA TTC CIG ICC ACA CCC TćT AAC Λ (Γ CCT TTC GCT AAC 22) ^

Ile Pro Clu GGu Cheese Leu Ile Ile ALa Gic A ^ Ua Ua Leu AIc a ^ sc

-25 -20 -15

TAC CCC AAC (TC? &lt;CC ^ AGA TAG ATT TAC CGA AGA CCC TAC ATATCAATA 255

Val ALa Met Met GGu Arg Cpu Gic Lys Arc Acc Pro Asa G ^ p Cys Lsl

-10 -5 15

CCC CCA CCA TTU? AAT Gd GTA CAT A7A CCC ACA CIA CIA CCA TACA dC 300

Clu Pro Pro Cheese Ua Gly IAo coc Lys sic Arc He How much Arc U? ehe

15 20

TAC GAC CGA ACC? &lt;GC? CCC TAC TCT (GAC ACA? TTC CCT TAC (GC¹ CCC CTCC 349

Tyr Asp Aa TAh. Ala Gly Ietu Cys Glu Uir Rie Val T / y Gly Gly cyy

30 35

AGA GCT AA AAC ACC AAC TTC AAC TAC? CCC CGA GAC TC AAC GAC ACC? 337

Arg Aa Asi Ag Asn An Fhe Lys Ser Aa Glu Ap Cyy Met Glu Ua

45 50

168 250

418

TCT GCT GGT GCC TAATCIAGA Cys Gly Gly ALa (2) INFORMATION ABOUT SEC IDENTIFICATION NUMBER 6:

(I) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 111 amino acids (3) TYPE: amino acid (D) TOFOLOGY: Hl ^^ C ^ wa (II) TYPE OF PARTICLE; protein (X1) SEQUENCE DESCRIPTION: IDENTIFICATION NUMBER 8:

Met Lys Ala VAL Ehe Lsu Val Leu Ser Lsu Ile Gly Ehe Cys Trp Aa,

-53 -50 -45 -40

Gln Pro Val Thr Gly Asp Glu Cheese Cheese VA_ Glu Ile Pro GLu Glu Cheese

-35 -30 -25

Lsu Ile Ile Ala Glu Asn Tir Thr Leu ALa Asn Val ALa Met Ala Glu

<td>Arg</td><td>Lsu</td><td>GLU</td><td>lys</td><td>Arg</td><td>Arg</td><td>Pro</td><td>Asp</td>

<td> -5</td><td></td><td></td><td></td><td></td><td> 1</td><td></td><td></td>

<td>Gly</td><td>Pro</td><td>Cys</td><td>lys</td><td>ALa</td><td>Arg</td><td>How much</td><td>How much</td>

<td></td><td></td><td></td><td> 15</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>Lsu</td><td>Cys</td><td>GLU</td><td>aurochs</td><td>phe</td><td>val</td><td>Tyr</td>

<td></td><td></td><td> 30</td><td></td><td></td><td></td><td></td><td> 35</td>

<td>own</td><td>phe</td><td>lys</td><td>Cheese</td><td>ALa</td><td>Glu</td><td>as-</td><td>Cys</td>

50 (2) SEQUENCE INFORMATION

Phe Cys Leu Glu Pro Pro Ser Thr 5 10

Arg Tyr Phe Tyr Asp ALa Thr Ala 20 25

Gy Gly Cys Arg ALa Asn Arg Asn 44

Met Gl · - Thr (Cy Gy Gy -Aa 55

ABOUT IDENTIFICATION NUMBER

9:

<td> (1)</td><td>charac (AND) (B) (C) (D)</td><td>SEQUENCE FIGURE: LENGTH: 418 pairs of TTF casades: Nucloinic acid NUMBER OF THREADS: one TOPOLOGY: Linear</td>

<td> (11)</td><td>TYPE</td><td>PARTICLES: cDNA</td>

<td>(V1)</td><td>SOURCE (AND)</td><td>ORIGINAL: ORGANISM: synthetic</td>

<td>(1X)</td><td>FEATURE: (AND) (B)</td><td>NAME / KEY: CDS LOCATION: 77..409</td>

168 250

<td>(1X)</td><td>FEATURE: (AND) (B)</td><td>NAME / KEY: LOCATION:</td><td>syg peptide 77..235</td>

<td>(1X)</td><td>FEATURE: (AND)</td><td>NAME / KEY:</td><td>mat peptide</td>

<td></td><td>(E)</td><td>LOCATION:</td><td> 236.,409</td>

(X1) SEQUENCE DESCRIPTION: IDENTIFICATION NUMBER 9:

GAmATT JAAGAATAGΓ TCAmAACA AGAnACAAA CmAAFrT JaTAJAJAAΓ 60

AIAAAJGAJJ AAAAGA ATG AAG GCT GTT TTC TTC GIT TTC TCC TTG AU 109

Mat Lys Ala Val Phe Leu Val leu Ser Leu He

-53 -50 -45

GGA TTC TGC TTG! GC CAA ΟΓ, GTC ACT GGC GA GAA TTCA TC? (GIT GAG 157

Gly Phe Cys Τρ Ha Gln Eto VH Tut Gly Ηρ Glu Ser- Ser Val Glu

-40 -35 -30

ATT CCG GAA GA GCT CG AAG AU GCT GAA MC AU AAC HG CGT MC 205

Ile Pro Glu Glu Cheese Leu Ile Ile Ala Glu Asn Tr Thr leu Ha ten

-25 -20 -15

GTC GCC ATC GCT GAG ACH TG dG MA AGA AG! CCC? CGT HERE? TG 223

Val Ala Mat; Ha Glu Hg leu du Lys Hg Hg Eto Hp EPie CCS Ilu

-10 -5 1 *5

GAA CCT CCA TCT ACT dG CCA TTG AA (U TAA AAG TAG TG EA TG 301

Glu Pro Pro Ser Ttrr dy Eto O / s L ^ s Ha Hg Ile He leu Tyy EPie ”5 20

TAC AAC GCC AAG GCT GCT TT! ICT CCA ACT TG CTT UA GCT GGG TTG 344

Tyr Asn Ha Lys Ala CTy Leu Ccs GGn ITr Phr VH Tyr dy dy CCy '30 35 *

AGA GGT AAC GGT AAC CCA TG UA ITT GCT GGA GGA TTG AAG AAG ACT 339

Arg dy Hn dy Asn dn EPie Tyr See Ha du Asp Ccs Met Arg ITr

44 55

TCT GGT GGT GCC TArTCTrAr 418

Quiet Gly Ha

INFORMATION ON THE SEQUENCE ABOUT IDEF YF ik

KAC U

NYM 10:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 111 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (11) PARTICLE TYPE: protein

168 250 (X1) DESCRIPTION OF THE SEQUENCE: SEQUENCE WITH THE IDENTIFICATION NUMBER 10:

<td>Underworld -53</td><td>lys</td><td>ALa</td><td>vai -50</td><td>Fhe Leu</td><td>vai</td><td>Leu</td><td>Cheese -45</td><td>Leu</td><td>How much</td><td>Gly Fhe Cys -40</td><td>Trp Ala</td>

<td>Gln</td><td>Pro</td><td>val -35</td><td>Thr</td><td>Gly Asp</td><td>Glu</td><td>Cheese -thirty</td><td>Cheese</td><td>val</td><td>Glu</td><td>Ile Pro Glu -25</td><td>Glu Cheese</td>

<td>Leu</td><td>How much -twenty</td><td>Ilee</td><td>aa</td><td>Glu Aah</td><td>(Tr -15</td><td>TTu-</td><td>Ileu</td><td>-aa</td><td>, An</td><td>Val Aa Met -10</td><td>ALa Gu</td>

<td>Arg -5</td><td>Leu</td><td>CG'l</td><td colspan="2">Lys Ag Arj 1</td><td>Pro</td><td>ap</td><td>Rie</td><td>CCS 5</td><td>leu</td><td>Gu Pro Pro</td><td>Ser 'Thr 10</td>

<td>Gly</td><td>Pro</td><td>Cys</td><td>lys 15</td><td>ALa Arg</td><td>How much</td><td>How much</td><td>Leu twenty</td><td>Tyr</td><td>phe</td><td>Tire Asn ALa 25</td><td>Lys ALa</td>

<td>Gly</td><td>Leu</td><td>Cys thirty</td><td>Gln</td><td>Thr phe</td><td>val</td><td colspan="5">Tyr Gly GLy Cys Arg Gy Asn 33 40</td><td>Gy Asn</td>

<td>Gln</td><td>phe</td><td>Tyr</td><td>sst</td><td>tAl Gu</td><td>TAP</td><td colspan="5">Cyy Met tAg TTr tCy GGyGly</td><td>aa</td>

50 55 (2) SEQUENCE IDENTIFICATION NUMBER 11 (1) SEQUENCE CHARACTERISTICS:

<td rowspan="2"></td><td rowspan="2">(AND) (3) CC) (D)</td><td colspan="2">LENGTH: 508 base pairs TYPE. nucleic acid</td>

<td>NUMBER OF THREADS: TOPOLOGY: L.</td><td>one regimes</td>

<td> (11)</td><td>TYPE</td><td colspan="2">PARTICLES: cDNA</td>

<td>(Vi)</td><td>SOURCE (AND)</td><td colspan="2">ORIGINAL: ORGANISM: synthetic</td>

<td>(1X)</td><td>.FEATURE:' (A) - (B)</td><td>NAME / KEY: LOCATION:</td><td>CDS 77..499</td>

<td>(IX)</td><td>FEATURE: kA) (B)</td><td>NAME / KEY: LOCATION</td><td>syg peptide: 77..331</td>

<td>(1X)</td><td>FEATURE: (AND) (3)</td><td>NAME / KEY: LOCATION:</td><td>mat_peptyd 332..499</td>

(X1) SEQUENCE DESCRIPTION: SEQUENCE. ABOUT IDENTIFICATION NUMBER 11:

GAATTCCATT CAAGAATAGT TCAAACAAGA AGAITA ^ CAAA CTATCAATIT CATACACAAT

168 250

ACACAOGCTT CCCAGC TTC TGT TCT CCC? TCT ATT ITT TCT GCC GTC TT lOi

Met Arg Phr Pro Ser Ile Pre Thr Tla Val Ten

<td></td><td> -85</td><td> -80</td><td> -75</td>

<td>TTC GCC GCC TKK</td><td>TCC CGC TTA GCT GCT</td><td>CCC CGTC AC ACC</td><td>¹ ACC ACC (GCT 1157</td>

<td>PTe Ca Ma See</td><td>See JAit Teu Ca Ca -70</td><td>Ero Vh1 An TCr -65</td><td>Tr-Tr Glu -60</td>

<td>GTT GCC ΤΜ GCC</td><td>KCA ATT ΚΜ GCT GCT</td><td>GCT GTC ACC GGT</td><td>TAC CCC GTC 205</td>

<td>Aso du Tr Ca '-55</td><td>dn eiP orc A1g uiA -50</td><td colspan="2">Ca VaI Ulu GIt Tyr S ec Ap -45</td>

<td>CTA GAC GGG GCT</td><td>CCC GCT HT GCT GCC</td><td>CCC CCC CCT CCC</td><td>CCC CGC CCC 255</td>

<td>Leu du dy Asp -40</td><td>Fre Asp Val Ala Val -35</td><td>Leu Pro Pre Ser -30</td><td>Asn Ser Crr</td>

<td>ACC TAC GGG ICC.</td><td>TCC TCC ATT CCC ACT</td><td>CCC ACC GGC AAG</td><td>ACC GGC GGC 331</td>

<td>Asn Asn dy Ilu -25</td><td>Ilu Phr Ile Asn Thr -20</td><td>Thr Ile Ca Sn -15</td><td>Ile Ca Ca</td>

<td>TCA GAC GCT GGG</td><td colspan="2">GIC CCC TTK GCT CCC CGC GAT TTK TCC</td><td>CCC GAC CCC 339</td>

<td>Lys du du Gly -10</td><td colspan="2">Val Ser Leu Asp Lys Arg Asp PTe Cys -5 1</td><td>Leu du Pro 5</td>

<td>CCC TAC AKT Gd</td><td>CCC TGC CCT GC CGC</td><td>CCC CCC AGA CAC</td><td>CCC CCC CCC 397</td>

<td>Pro Tyr Tri dy 10</td><td>Pro Cys Lys ALa Arg 15</td><td>Ile Ile Arg Tyr</td><td>PTe Tyr Asn twenty</td>

<td>GCC CAG GCT GGC</td><td>ttk ccc ccc act πο</td><td>GTC CAC GGC GGC</td><td>CGC TGC GCT 445</td>

<td>ALa Lys ALa dy 25</td><td>Leu Cys Gin Trr Phe 30</td><td>Val Tyr dy Gly 35</td><td>Cys Arg ALa</td>

<td>TAG CCC CAC ACC</td><td>CCC GCT TCT GCT GCT</td><td>GCC CCC CCC AGC</td><td>ACC CCC GGT 493</td>

<td>Lys Ser Asn A = n</td><td>PTe du Cheese ALa du</td><td>Csp Cys Met Arg</td><td>Crr Cys dy</td>

44 50

GGC GCC TTAiaTGA 508

Gly Ca (2) SEQUENCE IDENTIFICATION NUMBER 12:

(I) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 141 amino acids (B) TYF: amino acid (D) COFOLOGIT: linear (II) PARTICLE TYPE: Tiaiko (X1) SEQUENCE DESCRIPTION: IDENTIFICATION NUMBER SEQUENCE 12:

Met Arg PPr Pro Ser Ile Fhu Tr Ca Val Ja Le Ala Phe Sec Ser

-85 -80 -75 -70

Ala Leu ACl Ala Prc Vd. Cn Tr Bt: Tr GIt Me Glu Thr Ci Gin

-65 -60 -55

168 250

<td>How much</td><td>Pro</td><td>ala</td><td>Glu</td><td>ALa</td><td>val</td><td>How much</td><td>Gly</td><td>quoted</td><td>Cheese</td><td>Asp</td><td>Leu</td><td>Glu</td><td>Gly</td><td>Asp</td><td>phe</td>

<td></td><td></td><td></td><td> -50</td><td></td><td></td><td></td><td></td><td> -45</td><td></td><td></td><td></td><td></td><td> -40</td><td></td><td></td>

<td>Asp</td><td>Va!</td><td>ALa</td><td>vai</td><td>Leu</td><td>Pro</td><td>Fiie</td><td>Cheese</td><td>ΛΑΧ</td><td>: er</td><td>J.1TT</td><td>ΠΑί n</td><td>/ ΑΠ and</td><td>Gly</td><td>ILau</td><td>TA 1LTŁ</td>

<td></td><td></td><td> -35</td><td></td><td></td><td></td><td></td><td> —30</td><td></td><td></td><td></td><td></td><td> -25</td><td></td><td></td><td></td>

<td>fhe</td><td>How much</td><td>own</td><td>Tłnr</td><td>chr</td><td>How much</td><td>ALa</td><td>Cheese</td><td>IIe</td><td>AAa</td><td>AAa</td><td>lys</td><td>Gilu</td><td>games</td><td>gly</td><td>val</td>

<td></td><td> -20</td><td></td><td></td><td></td><td></td><td> -15</td><td></td><td></td><td></td><td></td><td> -10</td><td></td><td></td><td></td><td></td>

<td>Cheese</td><td>Leu</td><td>Asp</td><td>lys</td><td>ZAG</td><td>ap</td><td>Rhe</td><td>Cys</td><td>Leu</td><td>Gilu</td><td>poo</td><td>Pro</td><td>Tyr</td><td>TCh</td><td>gly</td><td>EPTO</td>

<td> -5</td><td></td><td></td><td></td><td></td><td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td>

<td>Cys</td><td>lys</td><td>ala</td><td>Arg</td><td>How much</td><td>How much</td><td>.ag</td><td>quoted</td><td>Phie</td><td>You</td><td>A = n</td><td>ala</td><td>lys</td><td>Zaa</td><td>gly</td><td>How many</td>

<td></td><td></td><td></td><td> 15</td><td></td><td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 22</td><td></td><td></td>

<td>Cys</td><td>Gin</td><td>Thr</td><td>phe</td><td>val</td><td>quoted</td><td>Gly</td><td>Gly</td><td>Cys</td><td>Aaj</td><td>ala</td><td>lys</td><td>Cheese</td><td>ain</td><td>own</td><td>phe</td>

<td></td><td></td><td> 30</td><td></td><td></td><td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td>

Hu Ser Ala GLu Asp Cys Mat Arg Tłcr Cyy Gli Gly Ala 45 50 55 (2) INFORMATION ABOUT SEQUENCE IDENTIFICATION NUMBER 13:

(I) CHARACTERISTICS OF SEEW ^ EN '] ^:

<td></td><td>(AND) (B) (C) (D)</td><td>LENGTH: 508 pao CYP bases: ILOIC THREAD nucleic acid: one TOPOLOGY: linear</td>

<td>(N)</td><td>TYPE</td><td>PARTICLES: cDNA</td>

<td>(V1)</td><td>SOURCE (AND)</td><td>ORIGINAL: ORGANISM: synthetic</td>

<td>(1X)</td><td>FEATURE: (AND) (B)</td><td>NAME / KEY: CDI LOCATION: 77..499</td>

<td>(1X)</td><td>C ^ T ^ A: (AND) (B)</td><td>NAME / KEY: syg_pepTyd LOCATION: 77..331</td>

<td>(IX)</td><td>FEATURE: (AND) (B)</td><td>NAME / KEY: mat_peptid LOCATION: 35ε ..- 477</td>

(X1) SEQUENCE DESCRIPTION: IDENTIFICATION NUMBER 13:

GAATCCTATT CAAAAAAAT CCAAACAAGG AAATACAAA TTATCAACT ΤΑΤΑΤΑΤΑΑΤ

ACAAGOAACC AAAAGA ATG AGA CCT CCT CCA ATT -TTT ATT GTA GTT TTA

Mat Arg Fhr Pro Ser Ile Ehe Thr Ala Va! Leu

-85 -80 -75

109

068 25°

TTC GTA GCA TCA TCAC &lt;GC TA CTT? CTCC CCA CTAC AAC CCT ACTA ACCC CGA

Ehe ALi ALi Ser S Aa Ilu Aa Aa Pro Val Asn The GAh TAh Glu

-70 -65 -60

CAT TAA ACG GCT. AA AAA CCO (CCA (GA CCC (GAC AAC GCA TAC TAA CCC

Asp Clu Anc Aa GIt Ile EPo Aa GGu Aa Vv1 Ile Cly Ί? / Sss Acp

-5Ó -50 -45

AAA GCA TCC CAT ATT GAT CCT &lt;GT? (TAC TAS CCA TAA TTC AAC ACCC AAA

Leu Tlu Cly A = p Phe Asp Vv1 Aa Vv1 Isei Pro Rte S ^ r An S &lt;^ Uhc

-40 -35 -30

AAT AAT TCC ATT TIT AAA AAA AAA? ACC¹ AAT .AAA (TCC ACCC .AAA CCC CTC

Asn Asn Tly Leu Leu Phe Ile Asn Uir Tr Ile Aa Cheese Ile Aa Aa

-25 -20 -15

AAA GCA CCC TTC TAA TCA TAA CCA? AA.A JAT. (CA? DAC TATA TAC CTAC CTT

Lys Tlu Tlu Cly Val Ssc Ilu Ap Lvs Ag Acs Re CCs Ilu Glu EPo

-10 -5 15

TCA TAC ACA GCA CCA ACA AAA CTT JAG AAC ACA AAA TAC dAC IAC AA

Pro Ayc Ahr Cly Pro Cys Lss Aa JAg LLe Ile Ag Tys Ehe 1? / An

15 20

CCC AAC GCA GCA TAC TCA CCA ACC dAC dA TAA GGT CTG TAA AAT CCC

Al Lys Aa Cly Leu Cys GCn TAm Hm Vv1 TAs Gly 'Gly CCs ^ r Aa

33 33

AAT ACC AAC AAT TAC GTA TAC CTC GTA GCA TAT ACA AAC ACC HAT GCT

Lys Ser Asn Asn Phe Al See Aa Glu Aa CCs MMe Acg TAr Cys Gly

44 55

CCA GTC TAATTAGA

Tly Aa 55

157

205

253

301

349

397

445

493

508 (2) INFORMATION ON SEQUENCE IDENTIFICATION NUMBER 14:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 141 amino acids (B) AYP: rhniac acid (Di TOPOLOGY: linear (n) TYPE OF PARTICLE: protein (X1) SEQUENCE DESCRIPTION: SEQUENCE D IDENTIFICATION NUMBER 14:

Met Arg Phe EPo Sss Ile Ehe ISt Aa Val Ubu Rv Aa Ap Sa- Ser “85 -80 -75 -77

Al Leu Al Aa EPo Val Asn Ttr Thr hSir Glu As? Sic Thc Aa Gin

-65 -60 -55

Ile Pro ALw Background Alw Val Ile Gly Asc Cheese Asp Lsu Glu Cly Asp Ohe -50 -45 -40

168 250

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu leu -35 -30 -25

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val -20 -15 -10

Cheese Leu Asp Lys Arg Asp Phe Cys Leu Glu Pro Pro Tyr Thr Gly Pro

-5 1 5 10

Cys Lys Ala Arg Ile Ile Arg Tyr Phe Tyr Asn Ala Lys Ala Gly Leu

20 25

Cys Gln Thr Phe Val Tyr Gly Gly Cys Arg Ala Lys Ser Asn Asn Phe 30 35 40

Ala Ser Ala Glu Asp Cys Met Arg Thr Cys Gly Gly Ala 45 50 55 (2) INFORMATION ON SEQUENCE IDENTIFICATION NUMBER l5:

(l) SEQUENCE CHARACTERISTICS.

(A) LENGTH: 412 base pairs (B) TYPE: nucleic acid (C) QUANTITY THREAD: one (D) TOPOLOGY: linear (li) TYPE OF PARTICLE: cDNA (vi) ORIGINAL SOURCE:

<td></td><td>(AND)</td><td>ORGANISM: sy</td><td>ntetyczna</td>

<td>(IX)</td><td>C ύ CHA: (A)</td><td>NAME / KEY:</td><td>CDS</td>

<td></td><td>(B)</td><td>LOCATION:</td><td> 77..403</td>

<td>(Ix)</td><td>FEATURE: (AND)</td><td>NAME / KEY:</td><td>syg_peptyd</td>

<td></td><td>(B)</td><td>LOCATION</td><td> : 77..235</td>

<td>(Ix)</td><td>FEATURE: (AND)</td><td>NAME / KEY:</td><td>mat peptide</td>

<td></td><td>(B)</td><td>Location:</td><td> : 236..403</td>

(XI) DESCRIPTION OF THE SEQUENCE: SEQUENCE WITH THE IDENTIFICATION NUMBER 15:

GAATTCCATT CAAGAATAGT TCAAACAAGA AGATTACAAA CTATCAATTT CAIACACAAT

ATAAAOGACC AAAAGA ATC AAG GCT GTT TTC TTC GTT TTC TCC TTC ATC Met Lys Ala Val Phe Leu Val Leu Ser Leu Ile -53 -50 —45

GGA TTC TCC TCG GCC CAA CCA GTC ACT GGC GAT GAA TCA TCT GTT GAG Gly Phe Cys Trp Ala Gln Pro Val Thr Gly Asp Glu Cheese Val Glu

-40 -35 -30

109

157

168 250

<td colspan="2">ATT CCG GAA GAG TCT</td><td rowspan="2">CTC Leu</td><td colspan="6">ATC ATC GCT GAA AAC ACC ACT TTC GCT AAC</td><td rowspan="2"> 205</td>

<td>Ile Pro Glu -25</td><td>Glu Cheese</td><td>How much -twenty</td><td>How much Ala Glu</td><td>Asn Ihr -15</td><td>Ihr</td><td>Leu</td><td>Ala Asn</td>

<td>GTC GCC ATS</td><td>GCT GAG</td><td>AGA</td><td>TTC</td><td>GAG AAG AGG</td><td>GAT TTC</td><td>TCT</td><td>TIG</td><td>GAA CCT</td><td> 253</td>

<td>Val Ala Met</td><td>Ala Glu</td><td>Arg</td><td>Leu</td><td colspan="2">Glu Lys Arg Asp Fhe</td><td>Cys</td><td>Leu</td><td>Glu Pro</td><td></td>

<td> -10</td><td></td><td> -5</td><td></td><td></td><td> 1</td><td></td><td></td><td> 5</td><td></td>

<td>CCA TCT ACT</td><td>GCT CCA</td><td>TCT</td><td>AAA</td><td>GCT AGA ATC</td><td>ATC AGA</td><td>TAC</td><td>TTC</td><td>TAC GAC</td><td> 301</td>

<td>Pro Ser Ihr</td><td>Gly Pro</td><td>Cys</td><td>lys</td><td>Ala Arg Ile</td><td colspan="2">Ile Arg Tyr</td><td>phe</td><td>iyr Asp</td><td></td>

<td></td><td> 10</td><td></td><td></td><td> 15</td><td></td><td></td><td> 20</td><td></td><td></td>

<td>GCC ACT GCT</td><td>GCT TTC</td><td>TCT</td><td>GAA</td><td>ACT TTC CTT</td><td>TAC GCT</td><td>GGC</td><td>TCC</td><td>AGA GCT</td><td> 349</td>

<td>Ala Itr Ala</td><td>Gly Leu</td><td>Cys</td><td>Glu</td><td>Ihr Phe Val</td><td colspan="2">Tyr Gly Gly</td><td colspan="2">Cys Arg Ala</td><td></td>

<td> 25</td><td></td><td></td><td></td><td> 30</td><td></td><td> 35</td><td></td><td></td><td></td>

<td>AAC AGA AAC</td><td>AAC TTC</td><td>AAG</td><td>TCT</td><td>GCT GAA GAC</td><td>TCC ATC</td><td>GAA</td><td colspan="2">ACT TCT GCT</td><td> 397</td>

<td>Asn Arg Asn</td><td>Asn Phe</td><td>lys</td><td colspan="2">Ala Glu Asp</td><td>Cys Met</td><td>Glu</td><td colspan="2">Ihr Cys Gly</td><td></td>

<td> 40</td><td></td><td></td><td> 45</td><td></td><td> 50</td><td></td><td></td><td></td><td></td>

<td colspan="2">GGT GCC TAATCIAGA</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 412</td>

<td>Gly Ala</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 55</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

(2 'INFORMATION ON SEQUENCE IDENTIFICATION NUMBER 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 109 amino acids tB) TYPE: Amino acid (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: protein (xi) SEQUENCE DESCRIPTION: IDENTIFICATION NUMBER SEQUENCE 16:

<td>Underworld</td><td>lys</td><td>ala</td><td>val</td><td>phe</td><td>leu</td><td>val</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>How much</td><td>Gly</td><td>phe</td><td>Cys</td><td>Trp</td><td>ala</td>

<td> -53</td><td></td><td></td><td> -50</td><td></td><td></td><td></td><td></td><td> -45</td><td></td><td></td><td></td><td></td><td> -40</td><td></td><td></td>

<td>Gin</td><td>Pro</td><td>val</td><td>Ihr</td><td>Gly</td><td>Asp</td><td>Glu</td><td>Cheese</td><td>Cheese</td><td>val</td><td>Glu</td><td>How much</td><td>Pro</td><td>Glu</td><td>Glu</td><td>Cheese</td>

<td></td><td></td><td> -35</td><td></td><td></td><td></td><td></td><td> -30</td><td></td><td></td><td></td><td></td><td> -25</td><td></td><td></td><td></td>

<td>Leu</td><td>How much</td><td>How much</td><td>ala</td><td>Glu</td><td>own</td><td>Ihr</td><td>Ihr</td><td>leu</td><td>ala</td><td>own</td><td>val</td><td>ala</td><td>Underworld</td><td>ala</td><td>Glu</td>

<td></td><td> -20</td><td></td><td></td><td></td><td></td><td> -15</td><td></td><td></td><td></td><td></td><td> -10</td><td></td><td></td><td></td><td></td>

<td>Arg</td><td>Leu</td><td>Glu</td><td>lys</td><td>Arg</td><td>Asp</td><td>phe</td><td>Cys</td><td>leu</td><td>Glu</td><td>Pro</td><td>Pro</td><td>Cheese</td><td>Ihr</td><td>Gly</td><td>Pro</td>

<td> -5</td><td></td><td></td><td></td><td></td><td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td>

<td rowspan="2">Cys</td><td>lys</td><td>ala</td><td>Arg</td><td>How much</td><td>How much</td><td>Arg</td><td>Tyr</td><td>phe</td><td>iyr</td><td>Asp</td><td>ala</td><td>Ihr</td><td>ala</td><td>Gly</td><td>leu</td>

<td></td><td></td><td> 15</td><td></td><td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td>

<td>Cys</td><td>Glu</td><td>Ihr</td><td>fhe</td><td>val</td><td>Tyr</td><td>Gly</td><td>Gly</td><td>cys</td><td>Arg</td><td>ala</td><td>own</td><td>Arg</td><td>own</td><td>own</td><td>phe</td>

<td></td><td></td><td> 30</td><td></td><td></td><td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td>

<td rowspan="2">lys</td><td>Cheese</td><td>ala</td><td>Glu</td><td>Asp</td><td>Cys</td><td>Underworld</td><td>Glu</td><td>Ihr</td><td>Cys</td><td>Gly</td><td>Gly</td><td>ala</td><td></td><td></td><td></td>

<td> 45</td><td></td><td></td><td></td><td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td>

168 250 (2) INFORMATION Ο

SEQUENCE Ο IDENTIFICATION NUMBER

17:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 508 base pairs (B) TYPE: nucleic acid (C) NUMBER OF THREADS: one (D) TOPOLOGY: Contract (ii)

TYPE OF PARTICLE: cuNA (VI)

ORIGINAL SOURCE:

(A) ORGANISM: synthetic (A) NAME / KEY: CDS (B) LOCATION: 77..499 (A) NAME / KEY: syg_peptide IB) LOCATION: 77..331 (ix) FEATURE:

(A) NAME / KEY: mar_peptid lB) LOCATION: 332..499 (XI) DESCRIPTION OF THE SEQUENCE: SEQUENCE WITH THE IDENTIFICATION NUMBER 17:

GAATTCCATT CAAGAAIAGT TCAAACAAGA AGATTACAAA CTATCAATTT CAIACACAAT 60

ATAAAOGATT AAAAGA ATG AGA TTT CCT TCA ATT TTT ACT GCA GIT TTA 109

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu -85 -80 -75

<td>TTC</td><td>GCA</td><td>GCA</td><td>TCC</td><td>TCC</td><td>GCA</td><td colspan="2">TTA GCT</td><td>GCT</td><td colspan="2">CCA GTC AAC</td><td>ACT ACA</td><td>ACA</td><td>GAA</td><td> 157</td>

<td>phe</td><td>ala</td><td>ala</td><td>Cheese</td><td>Cheese -70</td><td>ala</td><td>Leu</td><td>ala</td><td>ala</td><td>Pro Val -65</td><td>own</td><td>Thr thr</td><td>Thr -60</td><td>Glu</td><td></td>

<td>GAT</td><td>GAA</td><td>ACG</td><td>GCA</td><td>CAA</td><td>ATT</td><td>CCG</td><td>GCT</td><td>GAA</td><td>GCT GTC</td><td>ATC</td><td>GGT TAC</td><td>TCA</td><td>GAT</td><td> 205</td>

<td>Asp</td><td>Glu</td><td>Thr</td><td colspan="2">Ala Gin -55</td><td>How much</td><td>Pro</td><td>ala</td><td>Glu -50</td><td>Ala Vai</td><td>How much</td><td>Gly Tyr -45</td><td>Cheese</td><td>Asp</td><td></td>

<td>TTA</td><td>GAA</td><td>GGG</td><td>GAT</td><td>TTC</td><td>GAT</td><td>GTT</td><td>GCT</td><td>GTT</td><td>TTC CCA</td><td>TTT</td><td>TCC AAC</td><td>AGC</td><td>ACA</td><td> 253</td>

<td>leu</td><td>Glu</td><td colspan="2">Gly Asp -40</td><td>phe</td><td>Asp</td><td>val</td><td>ala -35</td><td>val</td><td>Leu Pro</td><td>phe</td><td>Asn cheese -30</td><td>Cheese</td><td>Thr</td><td></td>

<td>AAT</td><td>AAC</td><td>GGG</td><td>TTA</td><td>TTG</td><td>TTT</td><td>ΑΤΆ</td><td>AAT</td><td>ACT</td><td>ACT ATT</td><td>GCC</td><td>AGC ATT</td><td>GCT</td><td>GCT</td><td> 301</td>

<td>own</td><td>own -25</td><td>Gly</td><td>Leu</td><td>leu</td><td>phe</td><td>How much -twenty</td><td>own</td><td>Thr</td><td>Thr Ile</td><td>ala -15</td><td>Ile cheese</td><td>ala</td><td>ala</td><td></td>

<td>AAA</td><td>GAA</td><td>GAA</td><td>GGG</td><td>GTA</td><td>TCT</td><td>TTG</td><td>GAT</td><td>AAA</td><td>AGA GAT</td><td>TTC</td><td>TCT TTC</td><td>GAA</td><td>CCT</td><td> 349</td>

<td>lys -10</td><td>Glu</td><td>Glu</td><td>Gly</td><td>val</td><td>Cheese -5</td><td>Leu</td><td>Asp</td><td colspan="2">Lys Arg Asp 1</td><td>phe</td><td>Leo's cys</td><td>Glu 5</td><td>Pro</td><td></td>

168 250

CCC TAC CCT Gd CCCA TCT AAA GCT AGA ATC ATC- JAGA TC TTC TC CC &lt;dC

Pro Tyr Thr Gly JPro Cs Lys Ala Arg Ile Ile Ag Ty Rie Ts TAp

15 20

GCC ACT GCT GGT TTC TG (GAC CCT TTC GTT TAC CGG¹ CGG? TCC AGA (GC

ALa Thr ALa Gly Au C^s Glu Thr Phie Val Tyr Gly Gly Ccs Ag Aa

30 35

AAG AGA AAC CCC ΊΤΤΓ Al ITT? GCT GAA GAC TGC AT! GA ACT PTC CGG

Lys Arg Asn Asn LF.e Lvs Sm Ala Hu Asp Cys MmS du Tm CCs Gly

45 50 ggt Gyy tctcagc

GLy ALa 55 (2) INFORMATION ON THE SEQUENCE WITH THE IDENTIFICATION NUMBER (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 141 amino acids (B) TYPE: amino acid (D) TOPOLOGY: Linear (11) EODZAj KIESTECI KI; białoo

977

445

493

508

18:

(X1) DESCRIPTION OF THE SEQUENCE: SEQUENCE ABOUT IDENTIFICATION NUMBER 18:

Underworld

ALa

How much

Asp

ehe

Cheese

Cys

Cy^s

lys

Arg Phe IPo Sst Ile Phe Thr Aa Val Leu Rie Aa Ala Sm Sss -80 “55 -70

Leu ALa Aa Pro Val -Asn Thr Thr Thr Glu Asp Glu Tm Aa Gln -65 -60 -55

<td>Pro</td><td>ALa</td><td>du</td><td>ALa</td><td>val</td><td>How much</td><td>Gly</td><td>Tyr</td><td>Cheese</td><td>Asp</td><td>Leu</td><td>du</td><td>dy</td><td>Asp</td><td>phe</td>

<td></td><td></td><td> -50</td><td></td><td></td><td></td><td></td><td> -45</td><td></td><td></td><td></td><td></td><td> -4(0</td><td></td><td></td>

<td>val</td><td>aa</td><td>Vaa</td><td>leu</td><td>Pro</td><td>ROIs</td><td>Cheese</td><td>ass</td><td>Sec</td><td>tir</td><td>own</td><td>asi</td><td>Gly</td><td>Leu</td><td>au</td>

<td></td><td> -35</td><td></td><td></td><td></td><td></td><td> •330</td><td></td><td></td><td></td><td></td><td> -25</td><td></td><td></td><td></td>

<td>How much</td><td>TAs</td><td>PTU</td><td>htt</td><td>How much</td><td>ALa</td><td>Cheese</td><td>fox</td><td>ala</td><td>aa</td><td>Lyc</td><td>gic</td><td>Glu</td><td>Gly</td><td>Gau</td>

-20 -15 '-10

Leu Asp Lyy Aaj Asp EhP eC^s Leu Glu ito Pro You Tr Gly is&gt; 15 10

Lys Aa Arg Ile Ile Arg Tyr Phe Τ^τ Asp ALa Tm ALa Gly Leu 15 20 25

GLu PTr FFh W. Tyr Gly Gly cys Ag Aa Lyc Aia asn Aen Aie 30 55 40

ALa Glu Aa Cy cheese^s CSsM Glu Thr Cys Gly Gly Aa 45 50 55

168 250 (2) INFORMATION ABOUT SEQ ID No. 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH *. 412 pairs of zils ^ ić !.

(B) TYPE: nucleic acid (C) NUMBER OF THREADS: one (D) Linear TOPOLOGY

<td> (11)</td><td>TYPE</td><td>PARTICLES: cDNA</td>

<td>(Vi)</td><td>SOURCE (AND)</td><td>ORIGINAL: ORGANISM: synthetic</td>

<td>(Ix)</td><td>FEATURE: (AND) (B)</td><td>NAME / KEY: CDS LOCATION: 77..403</td>

<td>(Ix)</td><td>FEATURE: (AND) (B)</td><td>NAME / KEY :, sig ^ peptide LOCATION: 77..235</td>

<td>(Ix)</td><td>FEATURE: (AND) (B)</td><td>NAME / KEY: mat ^ peptide LOCATION: 243..404</td>

<td>^(X1)</td><td colspan="2">SEQUENCE DESCRIPTION: NUMBER SEQUENCE</td>

IDENTIFICATION 19:

GAATyccrTT JArGrryrly tcgaacaaaa αααπαεααα ttatjagttt catacacaat 60 ατααατιατε amasa atg aga gct gtt ttc ttg git ttg tcc ttg atc 109

Met Lys Ala VH Phe Leu VH Leu Ser Leu Ile

<td></td><td> -53</td><td> -50</td><td> -45</td>

<td>GGA</td><td>TTC TGC TTG! (GTC CCA</td><td>CCA GTC ACT GGT GAT</td><td>GAG TCC TCT MT (GAG 157</td>

<td>Gly</td><td>Phe Cys TT]? Ha gln</td><td>Pro Val Hit Gly Hp</td><td>Glu Cheese Cheese Val Glu</td>

<td></td><td> -40</td><td> -35</td><td>About 3 °</td>

<td>ATT</td><td>CJG GGA CUG TTC CJG</td><td>ATC ATC GCT GAA MC</td><td>ACCJ AAT TIG (GC? AAC 205</td>

<td>How much</td><td>Pro Glu Glu Cheese leu</td><td>Ile Ile Ha Glu Hn</td><td>Br ITr Leu Ha Hn</td>

<td></td><td> -25</td><td> -20</td><td> -15</td>

<td>GTC</td><td>GCC ATC GCT GAG AGA</td><td>TTG GAA AAA AAG GGA</td><td>ttg ogj itt; cga cct 253</td>

<td>val</td><td>Ha Met Ala Glu Aaj</td><td>Ilu Glu Lls 0aj Ars</td><td>Phe Cjs Ilu Glu EPo</td>

<td> -10</td><td> -5</td><td> 1</td><td> 5</td>

<td>CCA</td><td>TCT ACT GGT COC TCTT</td><td>AGA GAC AGA ATC AGC</td><td>AAG TTG ITT: TTG TAG 301</td>

<td>Pro</td><td>Ser Thr Gly Pro Cys</td><td>Lus Ha Arg Ile He</td><td>Arg iyr EPr TTs Hn</td>

<td></td><td> 10</td><td> 15</td><td> 20</td>

<td>GCC</td><td>AAA GCT GGT TTCTCT</td><td>TCA AAC TTCGGT TAG</td><td>GGT GGG ETC 0AA GGT 334</td>

<td>ala</td><td>Lys Ha Gly Leu Cyc</td><td colspan="2">GGi EG * Phe WL Tyr Gly Gly CJs Arg Gly</td>

<td></td><td> 25</td><td> 30</td><td> 35</td>

<td>AAC</td><td>GGC AAC AAC TTC AAG</td><td colspan="2">TCCGCT GM GAC TGC AAT GAA AAC EGC GGG 339</td>

<td>own</td><td>Gly Hn Asn Phe Lye</td><td colspan="2">Ala cheese. Glu Ar? Cys Met Glu EG * Cjs Gly</td>

<td></td><td> 40</td><td> 44</td><td> 50</td>

168 250

412

GGT GCC TAATCEAGA Gly Ala (2) SEQUENCE IDENTIFICATION NUMBER 20:

(I) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 109 amino acids (B) TYPE: Amino acid (D) TOPOLOGY: linear (II) PARTICLE TYPE: protein (XI) SEQUENCE DESCRIPTION: IDENTIFICATION NUMBER 20:

<td>Underworld</td><td rowspan="2">lys</td><td>ala</td><td>val</td><td>phe</td><td>Leu</td><td>val</td><td>Leu</td><td>Cheese</td><td>leu</td><td>How much</td><td>Gly</td><td>fhe</td><td>Cys</td><td>Trp</td><td>ala</td>

<td> -53</td><td></td><td> -50</td><td></td><td></td><td></td><td></td><td> -45</td><td></td><td></td><td></td><td></td><td> -40</td><td></td><td></td>

<td>Gin</td><td>Pro</td><td>val</td><td>Ihr</td><td rowspan="2">Gly</td><td>Asp</td><td>Glu</td><td>Cheese</td><td>Cheese</td><td>val</td><td>Glu</td><td>How much</td><td>Pro</td><td>Glu</td><td>Glu</td><td>Cheese</td>

<td></td><td></td><td> -35</td><td></td><td></td><td></td><td> -30</td><td></td><td></td><td></td><td></td><td> -25</td><td></td><td></td><td></td>

<td>Leu</td><td>How much</td><td>How much</td><td>ala</td><td>Glu</td><td>own</td><td>Thr</td><td>Thr</td><td>Leu</td><td>ala</td><td>own</td><td>val</td><td>ala</td><td>Underworld</td><td>ala</td><td>Glu</td>

<td></td><td> -20</td><td></td><td></td><td></td><td></td><td> -15</td><td></td><td></td><td></td><td></td><td> -10</td><td></td><td></td><td></td><td></td>

<td>Arg</td><td>Leu</td><td>Glu</td><td rowspan="2">lys</td><td>Arg</td><td>Asp</td><td>fhe</td><td>Cys</td><td>Leu</td><td>Glu</td><td>Pro</td><td>Pro</td><td>Cheese</td><td>Ihr</td><td>Gly</td><td>Pro</td>

<td> -5</td><td></td><td></td><td></td><td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td>

<td rowspan="2">Cys</td><td>lys</td><td>ala</td><td>Arg</td><td>How much</td><td>How much</td><td>Arg</td><td>iyr</td><td>phe</td><td>Tyr</td><td>own</td><td>ala</td><td>lys</td><td>ala</td><td>Gly</td><td>leu</td>

<td></td><td></td><td> '15</td><td></td><td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td>

<td rowspan="2">Cys</td><td>Gin</td><td>Thr</td><td>phe</td><td>val</td><td>Tyr</td><td>Gly</td><td>Gly</td><td>Cys</td><td>Arg</td><td>Gly</td><td>own</td><td>Gly</td><td>own</td><td>own</td><td>phe</td>

<td></td><td> 30</td><td></td><td></td><td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td>

<td>lys</td><td>Cheese</td><td>ala</td><td>Glu</td><td>Asp</td><td>Cys</td><td>Underworld</td><td>Glu</td><td>Ihr</td><td>Cys</td><td>dy</td><td>Gly</td><td>ala</td><td></td><td></td><td></td>

50 55 (2) INFORMATION ON SEQUENCE IDENTIFICATION NUMBER 21:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 508 base pairs (B) TYPE: nucleic acid (C) NUMBER OF THREADS: one (D) TOPOLOGY: linear

<td> (11)</td><td>TYPE</td><td>PARTICLES: cDNA</td>

<td>(VI)</td><td>SOURCE (AND)</td><td>ORIGINAL: ORGANISM: synthetic</td>

<td>(IX)</td><td>FEATURE: (AND) (B)</td><td>NAME / KEY: CDS LOCATION: 77..499</td>

168 250

<td>(1x)</td><td>FEATURE: (AND) (B)</td><td>NAME / KEY: LOCATION:</td><td>Signal-peptide 77331</td>

<td>(1X)</td><td>FEATURE: (AND)</td><td>NAME / KEY:</td><td></td>

<td></td><td>(B)</td><td>LOCATION:</td><td> 332. .477</td>

(X1) DESCRIPTION OF THE SEQUENCE: · SEQ ID No. 21:

GGACCCCATT CAAGCCTAGT CCAGGCACUC CCATGCAAA CTCTCACCCC CATACCCACT 60

AIACACGAIC CCACGA CTG AGA TC? CCT TC? ATT KAT ACT GCC dT CCC 109

Met Arg Rie Pro Ser Ile FT.e CTr Ala Val Lu

<td></td><td> -85</td><td> -80</td><td> -75</td>

<td>CCC GCC</td><td>GCC CCC CCC GOC CCC GGC GGC</td><td colspan="2">OKI CCC TAC AGC ACC? ACC GAC 115</td>

<td>Phe d</td><td>Ala Cheese Cheese Ala Ilu Ala Ala</td><td colspan="2">Pro Val An Άττ TCr Crr Glu</td>

<td></td><td> -70</td><td> -65</td><td> -60</td>

<td>GAC GCC</td><td>TCG GCGC ACA ATT (CC 11 Gl</td><td colspan="2">GC CGTC GTC GGT CCC CCC GAC 205</td>

<td>Asp Glu</td><td colspan="3">Crr Ala Gin Ile Pro Ma Glu Ma Val Ile Gly Tyr Cheese Asp</td>

<td></td><td> -55 -50</td><td></td><td> -45</td>

<td>CTA GCC</td><td>GGG GTC TTC (CCC CCT &lt;GK dT</td><td colspan="2">TCG CCC? TCT CCC AGC ACC ACC 253</td>

<td>Leu Glu</td><td>Gly As? Rie TAp Val TAa Val</td><td colspan="2">luu Pro EPie Ser An Ser Crr</td>

<td></td><td> -40 -35</td><td> -30</td><td></td>

<td>ATT ACC</td><td>GGG TUT ATT TCT TACA ATT ACT</td><td>AAC TGC CGC Ad</td><td>ATT CCT CCC 301</td>

<td>Asn Asn</td><td>dy Leu leu IKe He Asi ΊΤτ</td><td>ICr How much TCa Sn</td><td>How much ALa ALa</td>

<td> -25</td><td> -20</td><td> -15</td><td></td>

<td>ATT GCC</td><td colspan="2">GAC GCC GGA TTT! CGCGAAAACUCCGITC'TCr</td><td>TCG GA? OKAY? 339</td>

<td>Lys Glu</td><td colspan="2">du Glu Val Seu luu As -LUG Cg Aą PPr Cg</td><td>Lu Glu Pro</td>

<td>-H</td><td> -5</td><td> 1</td><td> 5</td>

<td>CCC CCC</td><td colspan="2">ACC GGG CCC. ICC TCA GGC AAG ATT ACC AAG TAC</td><td>TTC TAC TAC 337</td>

<td>Pro Tyr</td><td>Thr Glu Ppo Ccg Ly d Aa.</td><td>Ile Ile AAr Άτ</td><td>Rie You An</td>

<td></td><td> 10 15</td><td></td><td> 22</td>

<td>GCC AAG</td><td>GCT GGT CIG TCT TCA ACC TTC</td><td>cct tac: ccc cuuc</td><td>TGC ACA CCC 445</td>

<td>Ala Lys</td><td>Ala Gly Leu Cyc Gin Ucr Phe</td><td>Val TCg · Gly Gly</td><td>CKg Arg CLi</td>

<td></td><td> 25 30</td><td> 335</td><td></td>

<td>AAG CCC</td><td>AAC CAC CCC Ad CCT (CTT GAA</td><td>CCC TGC ATI (GAC</td><td>ACC? TGT Gd 443</td>

<td>Lys Ser</td><td>Asn Asn Phu Lys Cheese Ala Glu</td><td>Ap Cg MeU Glu</td><td>Tnr Cy Gly</td>

45 50

GGC GCC CCTTCCAGA 558

Gly Ala

168 250 (2) INFORMATION ON THE SEQUENCE WITH THE IDENTIFICATION NUMBER (i) CHARACTERISTICS OF THE SEQUENCE:

(A) DLUGOIC: 1 1U ίitΏlπoiów ^ .sow (B) TYPE: amino acid (D) TOPOLOGY: linj ^ c ^ wa (11) TYPE OF PARTICLE: protein (X1) SEQUENCE DESCRIPTION: IDENTIFICATION NUMBER SEQUENCE 22:

22:

<td rowspan="2">Mat Arg Fhe ryto -85</td><td rowspan="2">ier</td><td colspan="3">Ile Rre ΤΤοε Ala Val Leu Ehe Ala Ala Cheese</td>

<td> -80</td><td> -75</td><td> -70</td>

<td>ALa Leu A1a laA</td><td>pto</td><td>vil</td><td colspan="2">Aesn Tho Thir Thir Glu Aep Glu Thr ZAe. Gin</td>

<td></td><td> -65</td><td></td><td>-tzr \ 6 0</td><td> -55</td>

<td>Ilr Poo ZAa Gier</td><td>ala</td><td>VH</td><td>Ile Gly Cyr Sar Asp Leu Glu</td><td>Gly Asp Eh</td>

<td> -50</td><td></td><td></td><td> -45</td><td> -40 *</td>

<td>Asp Val ZAa Vćl1</td><td>TLU</td><td>eto</td><td>Ehe Ser Asn Gen Tar Tsn ZSsa</td><td>Gly Leu Elu</td>

<td> -35</td><td></td><td></td><td> -30 -25</td><td></td>

<td>Phr Ilr ZAn Thr</td><td>TCR</td><td>How much</td><td>.Ala Sal-Ue Ala Ala Aya Gli</td><td>Uli Giy WI</td>

<td> -20</td><td></td><td></td><td> -15 -10</td><td></td>

<td colspan="3">Ier Leu Ta? Lls Atg Aep</td><td>Ee Cys Ler Glu Ero Pro You</td><td>Thr Gly Eto</td>

<td> -5</td><td></td><td> 1</td><td> 5</td><td> 10</td>

<td>Cys Lys ZAa ZAg</td><td>How much</td><td>How much</td><td>Arg Cyr Er Tyr Asn ALa Lys</td><td>ALa Gly Ilu</td>

<td> 15</td><td></td><td></td><td> 20</td><td> 22</td>

<td>Cys Gin Hh Κτ</td><td>VVL</td><td colspan="2">Tyr Gly Giy Gys Tyg Alg Aya Seę-</td><td>Asa Ααπ Ehh</td>

<td> 30</td><td></td><td></td><td> 35 40</td><td></td>

<td>Lys Ier ZAe Giu</td><td>aa?</td><td colspan="2">Cys Met Glu Alu Cyr Tl? Gly GLSe</td><td></td>

<td> 45</td><td></td><td></td><td> 50 55</td><td></td>

<td colspan="2">(2) INFORMATION</td><td colspan="3">ABOUT IDENTIFICATION NUMBER SEQUENCE</td>

23:

(e) SEQUENCE CHARACTERISTICS:

<td></td><td>(AND) (B) (C) (D)</td><td>LENGTH: 508 base pairs TYPE: Nucleic acid NUMBER OF THREADS: one TOPOLOGY: linear</td>

<td> (11)</td><td>TYPE</td><td>PARTICLES: cDNA</td>

<td>(V1)</td><td>SOURCE (AND)</td><td>ORIGINAL: ORGANISM: synthetic</td>

<td>(1x)</td><td>FEATURE: (AND) (B)</td><td>NAME / KEY: CDS LOCATION: 77..499</td>

068 25°

<td rowspan="2">(Ix)</td><td rowspan="2">FEATURE: (AND) (B)</td><td colspan="2">NAME / KEY: syg_peptid</td>

<td>LOCATION:</td><td> 77..331</td>

<td>(IX)</td><td>FEATURE: (AND)</td><td>NAME / KEY:</td><td>mat-peptide.</td>

<td></td><td>(B)</td><td>LOCATION:</td><td> 332..499</td>

(XI) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 23:

GAAAASAAAA CAATAAAATA ACAAACTACA AGAAACAAA CATATAAAAA TATACTTCTT 60

ATACACCAJA AAAACA AAA AAC TAA CCT TA? ATA CAT ACT GCA Ad? ATT 109

Mat Arg Phe Pro Ser Ile Ehe Thr Ala Val Seau

-85 -80 -75

<td colspan="2">TAC GCA</td><td colspan="3">rcy Trr ta</td><td rowspan="3">GCA ala</td><td colspan="2">TAC? CAA GCT</td><td colspan="2" rowspan="2">ATA (CC AAC ACT TTC ACA AAA Pro VaL Asn Tir dn Thc GIt</td><td rowspan="3"> 157</td>

<td rowspan="2">Re</td><td rowspan="2">ALa</td><td rowspan="2">Jaa</td><td rowspan="2">Cheese</td><td rowspan="2">Sete -70</td><td rowspan="2">Leu Ala</td><td rowspan="2">ala</td>

<td> -65</td><td> -60</td>

<td>GAT</td><td>TAC</td><td>AOC</td><td>GTA</td><td>AAC</td><td>AAA</td><td>CCA GCT</td><td>GTA</td><td>GT?</td><td>GT? TCTC CTT TCC TAC CAT</td><td> 205</td>

<td>Asp</td><td>tlu</td><td colspan="2">Arr ALa</td><td>Tin</td><td>How much</td><td>Pro Ala</td><td colspan="3">Glu Ala W ile Cly Tyr Cheese Asp</td><td></td>

<td></td><td></td><td></td><td> -55</td><td></td><td></td><td></td><td> -50</td><td></td><td> -45</td><td></td>

<td>AAA</td><td>GCA</td><td>GCT</td><td>GAT</td><td>AAC</td><td>CAA</td><td>GIA GCA</td><td>CIA</td><td>ATT</td><td>ACA TTA ACA AAC CCC CCA</td><td> 253</td>

<td>Leu</td><td>tlu</td><td colspan="2">Cly Asp</td><td>rs</td><td>Asp</td><td>Val ALa</td><td>val</td><td>leu</td><td>Pro Re Ser Asn Ser Thr</td><td></td>

<td></td><td></td><td> -40</td><td></td><td></td><td></td><td> -35</td><td></td><td></td><td> —30</td><td></td>

<td>AAA</td><td>AAC</td><td>GCT</td><td>AIA</td><td>ATA</td><td>TAA</td><td>AAC AAT</td><td>ACCC</td><td>ACCC</td><td>ΑΠ 'ACCC ACCC AAA CCA TCA</td><td> 301</td>

<td>own</td><td>own</td><td>TLY</td><td>Leu</td><td>Lsu</td><td>Re</td><td>How much Asn</td><td>Hat</td><td>UA</td><td>Il (= JAa Cheese Ile ALa ALa</td><td></td>

<td></td><td> -25</td><td></td><td></td><td></td><td></td><td> -20</td><td></td><td></td><td> -15</td><td></td>

<td>AAA</td><td>GCA</td><td>TAC</td><td>CCC</td><td>TAA</td><td>TCA?</td><td>TAA GAT?</td><td colspan="2">TJAA ΑΒΑ!</td><td>AAT AAA? ATC AAA TAA ACA</td><td> 349</td>

<td>sys</td><td>Clu</td><td>Clu</td><td colspan="2">Cly Val</td><td>sro</td><td>Lsu A?</td><td colspan="3">Lys su? Aro Rr Cys Lsu Glu Pro</td><td></td>

<td> -10</td><td></td><td></td><td></td><td></td><td> -5</td><td></td><td></td><td></td><td> 1 5</td><td></td>

<td>CCA</td><td>TAA</td><td>ATA</td><td>CCC</td><td>CAA</td><td>give</td><td>TTAA ACC?</td><td>TCA.</td><td>CTC</td><td>ATC CAT. AC TAC AC AAC</td><td> 397</td>

<td>Pro</td><td>Tyr</td><td>Thr</td><td>Cly</td><td>Pro</td><td>Cys</td><td>Lys Alc</td><td>Ac?</td><td>His</td><td>Ile Jcg .Asc Re Άτ ^ n</td><td></td>

<td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td> 15</td><td></td><td> 22</td><td></td>

<td>GCA</td><td>ACC</td><td>TAA</td><td>GTA</td><td>AAA</td><td>ATA</td><td>CAA ATA</td><td>TAA</td><td>TTA</td><td>TAA GTC GTC TCA AGA GCA</td><td> 445</td>

<td>ala</td><td>sys</td><td>ALa</td><td>Gly</td><td>Leu</td><td>ays</td><td>GLn Anc</td><td>Re</td><td>val</td><td>Ayc Cis CLy Tys Acg ALa</td><td></td>

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

<td>AAC</td><td>GAC</td><td>ACA</td><td>ACC</td><td>AAT</td><td>AAG</td><td>CCT GCT?</td><td>GJG?</td><td>GAC?</td><td>TA cACAC CAA ATT TTA TCA</td><td> 493</td>

<td>sys</td><td>Clu</td><td>own</td><td>own</td><td>rs</td><td>lys</td><td>StS Ala</td><td>gic</td><td>AND?</td><td>Cys sMS Glu Arc Ays Cly</td><td></td>

45 00

CCA CCC TATACIACA 508

Gly All (2) SEQUENCE ID NUMBER 24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 041 amino acids

168 250 (B) TYPE: Amino acid (D) TOPOLOGY: Contract (11) PARTICLE TYPE: protein (XI) SEQUENCE DESCRIPTION: IDENTIFICATION NUMBER 24:

Met Arg Fhe Pro Cheese Ile Phe Thr Ala Val Leu Phe Ala Ala Cheese Ser

-85 -80 -75 -70

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gin —65 —60 —55

Ile Pro Ala Glu Ala Val Ile Gly Tyr Cheese Asp Leu Glu Gly Asp Ehe -50 -45 -40

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu -35 -30 -25

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val -20 -15 -10

Cheese Leu Asp Lys Arg Asp Phe Cys Leu Glu Pro ETO Tyr Thr Gly Pro

-5 1 5 10

Cys Lys Ala Arg Ile Ile Arg iyr Fhe Tyr Asn Ala Lys Ala Gly Leu

twenty 25 cys Gin Thr Phe Val iyr Gly Gly Cys Arg Ala Lys Glu Asn Asn Phe 30 35 40

Lys Ser Ala Glu Asp Cys Met Glu Thr Cys Gly Gly Ala 45 50 55 (2) INFORMATION ABOUT SEQUENCE IDENTIFICATION NUMBER 25:

(l) SEQUENCE CHARACTERISTICS:

<td></td><td>(AND)</td><td>LENGTH: 508 base pairs</td>

<td></td><td>(B)</td><td>TYPE: nucleic acid</td>

<td></td><td>(C)</td><td>NUMBER OF THREADS: one</td>

<td></td><td>(D)</td><td>TOPOLOGY: linear</td>

<td> (11)</td><td>TYPE</td><td>PARTICLES: cDNA</td>

<td>(VI)</td><td>SOURCE (AND)</td><td>ORIGINAL: ORGANISM: synthetic</td>

<td>(IX)</td><td>FEATURE: (AND) (B)</td><td>NAME / KEY: CDS LOCATION: 77..499</td>

<td>(IX)</td><td>FEATURE: (AND)</td><td>NAME / KEY: syg_peptid</td>

168 250 (B) LOCATION: 77..331 (IX) FEATURE:

(A) NAME / KEY-: mat_peptid (B) LOCATION. 332..499 (XI) DESCRIPTION OF THE SEQUENCE:. SEQUENCE WITH THE IDENTIFICATION NUMBER 25:

GAATTCCATT CAAGAAIAGT TCAAACAAGA AGATTACAAA CTATCAATTT CAIACACAAT 60

ATAAAOGATT AAAAGA ATG AGA TIT CCT TCA ATT TTT ACT GCA GTT TTA 109

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu -85 -80 -75

<td colspan="2">TTC GCA</td><td colspan="3">GCA TCC TCC GCA</td><td colspan="7">TTA GCT GCT CCA GIC AAC ACT ACA ACA GAA</td><td rowspan="2"> 157</td>

<td>phe</td><td>ala</td><td>Ala Ser</td><td>Cheese -70</td><td>ala</td><td>leu</td><td>ala</td><td>Ala Pro -65</td><td>val</td><td>own</td><td>Thr</td><td>Thr Thr Glu -60</td>

<td>GAT</td><td>GAA</td><td>ACG GCA</td><td>CAA</td><td>ATT</td><td>COG</td><td>GCT</td><td>GAA GCT</td><td>GTC</td><td>ATC</td><td>GCT</td><td>TAC TCA SPEC</td><td> 205</td>

<td>Asp</td><td>Glu</td><td>Thr Ala -55</td><td>Gin</td><td>How much</td><td>Pro</td><td>ala</td><td>Glu Ala -50</td><td>val</td><td>How much</td><td>Gly</td><td>iyr Cheese Asp -45</td><td></td>

<td>TTA</td><td>GAA</td><td>GGG GAT</td><td>TTC</td><td>GAT</td><td>CTT</td><td>GCT</td><td>CTT TTG</td><td>CCA</td><td>tir</td><td>TCC</td><td>AAC AGC ACA</td><td> 253</td>

<td>leu</td><td>Glu</td><td>Gly Asp -40</td><td>phe</td><td>Asp</td><td>val</td><td>ala -35</td><td>Val Leu</td><td>Pro</td><td>phe</td><td>Cheese -thirty</td><td>Asn Ser Thr</td><td></td>

<td>AAT</td><td>AAC</td><td>GGG TTA</td><td>TTG</td><td>TTT</td><td>ΑΤΆ</td><td>AAT</td><td>ACT ACT</td><td>ATT</td><td>GCC</td><td>AGC</td><td>ATT GCT GCT</td><td> 301</td>

<td>own</td><td>own -25</td><td>Gly Leu</td><td>Leu</td><td>phe</td><td>How much -twenty</td><td>own</td><td>Thr thr</td><td>How much</td><td>ala -15</td><td>Cheese</td><td>How much Ala Ala</td><td></td>

<td>AAA</td><td>GAA</td><td>GAA GGG</td><td>CTA</td><td>TCT</td><td>TTG</td><td>GAT</td><td>AAA AGA</td><td>GAT</td><td>TTC</td><td>TCT</td><td>TTG GAA CCT</td><td> 349</td>

<td>lys -10</td><td>Glu</td><td>Glu Gly</td><td>val</td><td>Cheese -5</td><td>leu</td><td>Asp</td><td colspan="2">Lys Arg Asp 1</td><td>phe</td><td>Cys</td><td>Leu Glu Pro 5</td><td></td>

<td>CCA</td><td>TAC</td><td>ACT GGT</td><td>CCA</td><td>TCT</td><td>AAA</td><td>GCT</td><td>AGA ATC</td><td>ATC</td><td>AGA</td><td>TAC</td><td>TTC TAC AAC</td><td> 397</td>

<td>Pro</td><td>Tyr</td><td>Thr Gly 10</td><td>Pro</td><td>Cys</td><td>lys</td><td>ala</td><td>Arg Ile 15</td><td>How much</td><td>Arg</td><td>Tyr</td><td>Phe Tyr Asn twenty</td><td></td>

<td>GCC</td><td>GAA</td><td>GCT GCT</td><td>TTG</td><td>TCT</td><td>CAA</td><td>ACT</td><td>TTC CTT</td><td>TAC</td><td>GCT</td><td>GGC</td><td>TGC AGA GCT</td><td> 445</td>

<td>ala</td><td>Glu</td><td>Ala Gly 25</td><td>Leu</td><td>Cys</td><td>Gin</td><td>Thr thirty</td><td>Fhe Val</td><td colspan="3">Tyr Gly Gly 35</td><td>cys Arg Ala</td><td></td>

<td>AAG</td><td>TCC</td><td>AAC AAC</td><td>TTC</td><td colspan="2">AAG TCT</td><td>GCT</td><td>GAA GAC</td><td colspan="2">TGC ATG</td><td>GAA</td><td>ACT TCT GCT</td><td> 493</td>

<td>lys</td><td>Cheese</td><td>Asn Asn</td><td>fhe</td><td colspan="2">Lys Ser</td><td>ala</td><td colspan="3">Glu Asp Cys Met</td><td>Glu</td><td>Thr Cys Gly</td><td></td>

45 50

GGT GCC TAATCIAGA Gly Ala (2) SEQUENCE IDENTIFICATION NUMBER 26:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 141 amino acids (B) TYPE: amino acid (D) TOPOLOGY: Linear (X1) SEQUENCE DESCRIPTION: IDENTIFICATION NUMBER 26:

<td rowspan="2">Mt Arg Rie -85</td><td rowspan="2">Pro</td><td rowspan="2">sm</td><td colspan="3">Ile Pie 'Th Aa VA Au Phe Aa Aa Sert Sm</td>

<td> -80</td><td> -75</td><td> -70</td>

<td>ALa Au Ma</td><td>aa</td><td>Pro</td><td>val</td><td>An Thr Thr Thr Glu Asp Glu Thr Ala</td><td>Gln</td>

<td></td><td></td><td> -65</td><td></td><td> -60 -55</td><td></td>

<td>How much Pro TAa</td><td>Glu</td><td>aa</td><td>val</td><td>Ile Gly Ty Sm Ap Au Glu Gly Ap</td><td>Rie</td>

<td></td><td> -50</td><td></td><td></td><td> -45 -40</td><td></td>

<td>Asp Val Aa</td><td>Vaa</td><td>au</td><td>Pro</td><td>Phe Ser An Sm Thr Asn An Gly Au</td><td>au</td>

<td> -35</td><td></td><td></td><td></td><td> -30 -25</td><td></td>

<td>Ehe He Acs</td><td>tr</td><td>aurochs</td><td>How much</td><td colspan="2">tAl Sse How many tAl tAl Lls Glu Glu Gly Val</td>

<td> -20</td><td></td><td></td><td></td><td> -10</td><td></td>

<td colspan="4">Leu Asp Lys Ag spp. Cheese</td><td>Rte cys Au Glu Mon Fri Ty Gly</td><td>Pro</td>

<td> -5</td><td></td><td></td><td> 1</td><td> 5 10</td><td></td>

<td>Cys Lys Al</td><td>Ag</td><td>How much</td><td>He</td><td>Arg Ty Rie Tc An Aa Glu Al Gly</td><td>au</td>

<td></td><td> 15</td><td></td><td></td><td> 20 25</td><td></td>

<td>Ccs Gln Tr</td><td>Peh</td><td>val</td><td colspan="2">Tsr Gly Gy CCy Arg Aa Lys Sse tAs tAs</td><td>FFh</td>

<td> 30</td><td></td><td></td><td></td><td> 35 40</td><td></td>

<td>Lys Ses Aa</td><td>Glu</td><td>Asp</td><td colspan="2">Cys Met Glu ICr CM Gly GTy As</td><td></td>

<td> 45</td><td></td><td></td><td></td><td> 55 55</td><td></td>

(£) SEQUENCE IDENTIFICATION WITH THE IDENTIFICATION NUMBER £ 7 (l) CHARACTERISTICS OF THE SEQUENCE;

<td></td><td>(C) (B) (C) (D)</td><td>LENGTH: 508 base pairs TYPE: Nucleic acid · NUMBER OF THREADS: one TOPOLOGY: linear</td>

<td>(Ii)</td><td>TYPE</td><td>PARTICLES: cDNA</td>

<td>(Vi)</td><td>ZZODŁO</td><td>ORYYlNALNE:</td>

<td></td><td>(C)</td><td>ORGANISM: synthetic</td>

<td>(Ix)</td><td>FEATURE. (AND) (B)</td><td>NAME / KEY: CDS LOCATION: 77..499</td>

<td>(and X)</td><td>FEATURE: (C) (B)</td><td>NAME / KEY: sygyepteptide LOCATION: 77..331</td>

<td>(Ix)</td><td>FEATURE: (C)</td><td>NAME / KEY: matjpeptide</td>

(B) LOCATION: 332..499 (xi) SEQUENCE DESCRIPTION: IDENTIFICATION NUMBER 2-7:

GACTTCTCTT TACGACTAGT TTTCACTACG TAGCTACMM CCTATTAMTT CTMATATAC 6 (0

MAAMOGCT CCACTC CCG TAG TCT CCT TCT iMC TCT? ACT¹ CGT CTT DCC 109

Mat: MA Ehe EPo See Ile Ehe TCh Ma Val Ilu

-85 -80 —75

<td rowspan="2">CCT phe</td><td colspan="2">GCC GT ITT</td><td colspan="8">CTT GTC TA GTT GTT TCC GTT ACC ACTT ACTC ACTC OM</td><td rowspan="2"> 157</td>

<td>MLi</td><td>It has S ^ r</td><td>Cheese -70</td><td>Ma Deu Ma</td><td>has</td><td>Ero -65</td><td>an</td><td>M Ήτ</td><td>Ώτ: -</td><td>TCh Gilu -60</td>

<td>GAC</td><td>GAA</td><td>ACTi GTC</td><td colspan="2">TAC MS? CTT GTT</td><td>GCC</td><td>GTT</td><td colspan="2">GCT MTT GCT</td><td>TAC</td><td>TTT. GA</td><td> 205</td>

<td>mp</td><td>hu</td><td>ΐττ Ma</td><td>dn</td><td>how many EPo Ma</td><td colspan="3">Glu Ma ad</td><td>He dy</td><td>and you</td><td>See Ms&gt;</td><td></td>

<td></td><td></td><td> -55</td><td></td><td></td><td> -50</td><td></td><td></td><td></td><td> -45</td><td></td><td></td>

<td>CIA</td><td>GAC</td><td>GGG (GA</td><td>TTC</td><td>CGC CTT GT</td><td>(GIT</td><td>TT</td><td>TTC</td><td>TT? TT</td><td>JMC</td><td>ACG ACT</td><td> 2^5</td>

<td>Leu</td><td>Glu</td><td>Gly Mp</td><td>ehe</td><td>JMp Vv1 Ma</td><td>Vc ^</td><td>leu</td><td>Pro</td><td>EPe Cheese</td><td>Mn</td><td>Ssu 0?</td><td></td>

<td></td><td></td><td> -40</td><td></td><td> -35</td><td></td><td></td><td></td><td> -30</td><td></td><td></td><td></td>

<td>MCC</td><td>MMC</td><td>HG TA</td><td>TTT</td><td>TTC MA CCT</td><td>ACT</td><td>MTT</td><td>ACC</td><td>gt: cgc</td><td>ACT</td><td>TCT GT?</td><td> 3101</td>

<td>Mn</td><td>Mn</td><td>dy Leu</td><td>Lu</td><td>Phe Ile Mn</td><td>cht</td><td>Thr</td><td>How much</td><td>Ma Cheese</td><td>How much</td><td>CLa Ma</td><td></td>

<td></td><td> -25</td><td></td><td></td><td> -20</td><td></td><td></td><td></td><td> -15</td><td></td><td></td><td></td>

<td>CAA</td><td>GAA</td><td>GAA TiT</td><td>DTC</td><td colspan="4">CTT Td GM MCC Ad GA?</td><td>TTC CGT</td><td>CTG</td><td>TAC CTT</td><td>3 ^^ S9</td>

<td>lys</td><td>Glu</td><td>a lot of</td><td>val</td><td colspan="4">Cheese leu Mp Lys Arg Mp</td><td>IP Cys</td><td>Leu</td><td>Glu EPo</td><td></td>

<td> -10</td><td></td><td></td><td></td><td> -5</td><td></td><td></td><td> 1</td><td></td><td></td><td> 5</td><td></td>

<td>CCC</td><td>TCC</td><td>ATT GCT</td><td>CTTC</td><td>CTC MCC GT?</td><td colspan="2">ACTA ATT</td><td>MT</td><td>MG TCC</td><td>TTT</td><td>TAC MAC?</td><td> 397</td>

<td>Pro</td><td>quoted</td><td>Cht Gly</td><td>RTO</td><td>T / s Lys Ma</td><td>mg</td><td>How much</td><td>How much</td><td>Pcg Ts-</td><td>ehe</td><td>Cit</td><td></td>

<td></td><td></td><td> 10</td><td></td><td></td><td> 15</td><td></td><td></td><td></td><td> 22</td><td></td><td></td>

<td>GCT</td><td>GAA</td><td>GTT HT</td><td>TT</td><td>CGT CTCc AAC</td><td>TTT</td><td>GIT</td><td>this:</td><td>di gg</td><td>tg:</td><td>AGA CGT</td><td> 445</td>

<td>ala</td><td>du</td><td>MLi dy</td><td>IYU</td><td>Ts dn TCh</td><td>ehe</td><td>val</td><td>"Τγ</td><td>dy Gly</td><td colspan="2">&lt;T^S Mg Ma</td><td></td>

<td></td><td></td><td> 25</td><td></td><td> 33</td><td></td><td></td><td></td><td> 35</td><td></td><td></td><td></td>

<td>CCT</td><td>GAA</td><td>CCT MMT</td><td>CTT</td><td>MC TT? GT</td><td>GA</td><td>GAT</td><td>TTT</td><td>AAC GAC</td><td colspan="2">ACC? Td GGT</td><td> 449</td>

<td>lys</td><td>du</td><td>Mn Mn</td><td>Ipe</td><td>See Mai</td><td>du</td><td>mp</td><td>&lt;Ts</td><td>MML Gilu</td><td colspan="2">'Th- &lt;You Gly</td><td></td>

<td></td><td> 40</td><td></td><td></td><td> 44</td><td></td><td></td><td></td><td> 50</td><td></td><td></td><td></td>

GGC GCC TAMTCCAGA 50S dy Ala (2) INFORMATION ABOUT SEQUENCE IDENTIFICATION NUMBER 28:

(I) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 141 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (II) PARTICLE TYPE: protein (Xl) SEQUENCE DESCRIPTION: IDENTIFICATION NUMBER 28:

<td colspan="4">Mat Arg Ełie EPro Ser Ile Fhe Ihr ALa leu He Ha ALa Cheese Ser</td>

<td> -85</td><td> -80</td><td> -75</td><td> -70</td>

<td>ALa</td><td colspan="2">Leu Ala Air Pro Vv1 Hn Gir Gir Tttro Alu Asa Glu Hr Ha</td><td>GGN</td>

<td></td><td> -65</td><td> -60 -55</td><td></td>

<td>How much</td><td colspan="2">Pro ALa GIa Air Ή Ile Gly Tyr Ser Aą leu Glu Gly Hp</td><td>phe</td>

<td></td><td> -50</td><td> -45 -40</td><td></td>

<td>Asp</td><td>Val ALa Val Ilu Pro Phe See</td><td>Hn Ssu ITr Hn Hn Gly Hu</td><td>hu</td>

<td></td><td> -35 —30</td><td> -25</td><td></td>

<td>Whew</td><td>Ile Asn Uh * Thr Ile Ala Ssu</td><td>How much AGa AAa Lus GAu GAu GGy</td><td>wi</td>

<td></td><td> -20 -15</td><td> -10</td><td></td>

<td>Cheese</td><td>Leu Asp Lye An grp BEe Thousands</td><td>Leu Alu Pro Pro Tyr Tir GGy</td><td>Pro</td>

<td> -5</td><td> 1</td><td> 5 11</td><td></td>

<td>cys</td><td>Lys ALa An Ιΐθ Ile Hg Ts *</td><td>Hee Tyr Asn Ala Alu Ala GGy</td><td>hu</td>

<td></td><td> 15</td><td> 20 25</td><td></td>

<td>Cys</td><td colspan="2">Gln Ger Fhe Val Tyr GGy Gly Cys An Ala Lys Glu 0An Hn</td><td>ehe</td>

<td></td><td> 30 35</td><td> 44</td><td></td>

<td>lys</td><td colspan="2">Cheese ALa GIa Aa? Cys; Mee Glu Ut * Cvs GIa. Gly Ma</td><td></td>

55 55 (2) INFORMATION ON THE SEQUENCE WITH THE IDENTIFICATION NUMBER 29 (l) CHARACTERISTICS OF THE SEQUENCE:

<td>(G)</td><td>LENGTH: 58</td><td>amino acids</td>

<td>(B)</td><td colspan="2">TYPE: amino acid</td>

<td>(D)</td><td>TOPOLOGY:</td><td>linoowa</td>

<td>(li) TYPE</td><td>PARTICLES:</td><td>protein</td>

(xi) DESCRIPTION OF THE SEQUENCE: SEQUENCE WITH THE IDENTIFICATION NUMBER 29:

Arg Eto Hp IF * e Cjs Lsu Alu Pro Pro Tyr Thr · Gly Pro Cys Lys Ha 15 10 15

Arg Ile Ile Hg Ty Rie Tyr Asn Ala Lys Ala Gly Leu Ty Gln ITr 20 25 30

Fhe VYl Gyr Gly G1g CJs Arg ALa Lys Grgo Asn Hn Phe Lys S ^ r AAa 35 40 45

Glu Asp Cys MeL · An The T /^s Gly Gly Ala 50 55

<img file="PL168250B1_D0001.tif" />

<img file="PL168250B1_D0002.tif" />

Fig. 2

Kidneys lgoda-Kidneys 3godi— Urine 3gode.

<img file="PL168250B1_D0003.tif" />

Fig. 3% of urine dose 20 τ-1-1-1-i-1-1-1-1-r

-3 -2 -1 0 1 2 3 4 5 6

Accident load

Fig. 4 η

thirty 20% of the dose in the kidneys after 3 hours fl

Ί-1-1-1-1-

70 80 90 100 denaturation temperature

Fig. 5

2.5 ί

1.5 1 accumulation rate in the kidneys

<img file="PL168250B1_D0004.tif" />

65 70 75 80 85 90 95 100 1 05 denaturation temperature

Fig. 6

5

MetAlaGluArgLeuGluLysArgGluProAspPheCysleuGluProProNcflI.

CATGGCTGAGAGATTGGAGAAGAGAGAGCCTGATTTCTGTTTGGAACCTCCACGACTCTTCAACCTCTTCTCTCTCGGACTAAAGACAAACCTTGGAGGT10 15 20 25

You rThrG 1 y ProS sLy s A1 aAr g Ilell eArgTyrPheTyrAs nA1aG1uAvaII

TACACTGGTCClATGTAAAGCTAGAATCATCAGATACTTCTACAACdCCGAAATGTGACCAGGTACATifrCGATCTTAGTAGTCTATGAAGATGTTGCGGCrS30 35 40

AlaGlyLeuCysGlnThrPheValTyrGlyGlyCysArgAlaGluArgAsngctggtttgtgtcaaactttcgtttacggtggciJgcagagctgaaagaaacCGACCAAACACAGTTTGAAAGCAAATGCCACCGACT 45 55

AsnPheGluSerAlaGluAspCysMetArgThrCysGlyGlyAlaStop

XbaI

AACTTCGAAljCTGCTGAAGACTGCATGAGAACTTGTGGTGGTGCCTAAT

TTGAAGCTTAGACG ^ TTCTGACGTACTCTTGAACACCACCACGGATTAGATC

Fig. 1

Department Department - Met— UP RP Circulation 90 copies

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