MXPA00011727A - Kunitz domain polypeptide and materials and methods for making it - Google Patents

Abstract

Proteinase inhibitors comprising a Kunitz domain are disclosed. The Kunitz domain comprises a sequence of amino acid residues as shown in SEQ ID NO:5, wherein the sequence is at least 90%identical to SEQ ID NO:2. Also disclosed are methods for making the proteinase inhibitors, and expression vectors and cultured cells that are useful within the methods. The proteinase inhibitors may be used as components of cell culture media, in protein purification, and in certain therapeutic and diagnostic applications.

Description

POLYPEPTIDE OF DOMICILE OF KÜNITZ AND MATERIALS AND METHODS FOR ITS ELABORATION BACKGROUND OF THE INVENTION In animals, proteinases are important in wound healing, destruction of extracellular matrix, tissue reorganization, and torrents that result in blood coagulation, fibrinolysis, and complement activation. Proteinases are released by inflammatory cells for the destruction of pathogens or external materials, and by normal and cancerous cells as they move through their surroundings.

The activity of proteinases is regulated by inhibitors; 10% of the proteins in the blood serum are proteinase inhibitors (Roberts et al., Critical Reviews in Eukaryotic Gene Expression (Critical Reviews in the Expression of the Eukaryotic Gene) 5: 385-436, 1995). A family of proteinase inhibitors, the Kunitz inhibitors, include trypsin inhibitors, chymotrypsin, elastase, kallikrein, plasmin, coagulation factors Xla and IXa, and cathepsin G. These inhibitors therefore regulate a variety of physiological processes, including the coagulation of blood, fibrinolysis, and inflammation.

Ref. 125392 Proteinase inhibitors regulate the proteolytic activity of target proteinases occupying the active site and thus preventing occupancy by normal substrates. Although the proteinase inhibitors fall into several unrelated structural classes, they all have an exposed loop (variously called an "inhibitor loop", a "reactive center", a "reactive site", or a "binding loop"). ) which is stabilized by intermolecular interactions between residues that are on the sides of the binding loop and the center of the protein (Bode and Huber, Eur. J. Biochem, 204: 433-451, 1992). The interaction between inhibitor and enzyme produces a stable complex that dissociates very slowly, releasing a virgin (undivided) inhibitor, or a modified inhibitor that is divided into the cleavage junction of the binding loop.

One class of proteinase inhibitors, the Kunitz inhibitors, are generally low molecular weight basic proteins that comprise one or more inhibitory domains ("Kunitz domains"). A Kunitz domain is a multiplicable domain of approximately 50-60 residues that form a central antiparallel beta sheet and a short terminal C helix. This characteristic domain comprises six cystine residues that form three disulfide bonds, resulting in a double-loop structure. Between the terminal region N and the first beta strand lies the active inhibitory binding loop. This bond is joined by disulfide via the P2 Cys residue with the hairpin loop formed between the last two beta strands. Kunitz domains isolated from a variety of proteinase inhibitors having inhibitory activity have been shown (eg, Petersen et al., Eur. J. Biochem., 125: 310-316, 1996; Wagner et al., Biochem. Biophyis. 186: 1138-1145, 1992; Dennis et al., J. Biol. Chem. 270: 25411-25417, 1995).

Proteinase inhibitors comprising one or more Kunitz domains include tissue factor pathway inhibitor (TFPI), tissue factor 2 pathway inhibitor (TFPI-2), precursor of β-amyloid protein (AβPP), aprotinin, and placenta bikunin. TFPI, an extrinsic pathway inhibitor and a natural anticoagulant, contains three Kunitz inhibitory domains linked together. The Kuni: z domain of amino terminal inhibits Vlla factor, plasmin, and cathepsin G; the second domain inhibits factor Xa, trypsin, and chymotrypsin; and the third domain has no known activity (Petersen et al., cited above). It has been shown that TFPI-2 is an inhibitor of the amidolitic and proteolytic activities of the human factor Vlla-tissue factor complex, factor Xla, plasma kalikrein, and plasmin (Sprecher et al., Proc. Na ti. Acad. Sci. USA 91: 3353-3357, 1994; Petersen et al., Biochem. 35: 266-272, 1996). The ability of TFPI-2 to inhibit factor Vlla-tissue factor complex and its relatively high levels of transcription in endothelial cells of umbilical vein, placenta and liver suggests a specialized role for this protein in hemostasis (Sprecher et al., Cited previously) . Aprotine (bovine pancreatic trypsin inhibitor) is a broad-spectrum Kunitz-type serine proteinase inhibitor that has been shown to prevent the activation of coagulation torrents. Aprotinin is a moderate inhibitor of plasma kallikrein and plasmin, and block fibrinolysis and extracorporeal coagulation has been detected in patients who were given aprotinin during open-heart surgery (Davis and Wittington, Drugs ^ 9: 954-983, 1995; Dietrich et al., Thorac, Cardiovasc Surg. 37: 92-98, 1989). Aprotinin has also been used in the treatment of septic shock, adult respiratory distress syndrome, acute pancreatitis, hemorrhagic shock, and other conditions (Westaby, Ann Thorac Surg 55: 1033-1041, 1993; Wachtfogel et al. J. Thorac, Cardiovasc, Surg. 106: 1-10, 1993). The clinical usefulness of aprotinin is believed to come from its inhibitory activity towards plasma kalikrein or plasmin (Dennis et al., Cited above). Placental bikunin is a serine proteinase inhibitor that contains two Kunitz domains (Delaria et al., J. Biol. Chem. 272: 12209-12214, 1997). Individual Kunitz domains have been expressed and shown to be potent inhibitors of trypsin, chymotrypsin, plasmin, factor Xla, and tissue and plasma kalikrein (Delaria et al., Cited above).

Known Kunitz-type inhibitors lack specificity and may have low potentiality. Lack of specificity may result in undesirable side effects, such as nephrotoxicity that occurs after repeated injections of high doses of aprotinin. These limitations can be overcome by preparing isolated Kunitz domains, which may have lower side effects than traditional anticoagulants. Accordingly, there is a need in the art for additional Kunitz-type proteinase inhibitors.

BRIEF DESCRIPTION OF THE INVENTION An object of the present invention is to provide novel Kunitz inhibitory proteins and compositions comprising the proteins. Another object of the invention is to provide materials and methods for the preparation of Kunitz inhibitory proteins. A further object of the invention is to provide antibodies that specifically bind to Kunitz inhibitory proteins.

In one aspect, the invention provides an isolated protein comprising a sequence of amino acid residues as shown in SEQ. NO. IDENTIFICATION: 5, where the sequence is at least 90% identical to residues 9 to 59 of SEC NO. IDENTIFICATION: 2 and where the protein has inhibitory activity of proteinase. In one embodiment, the protein is 51 to 81 amino acid residues in length. In other embodiments, the protein is 51 to 67 residues in length, preferably 55 to 62 residues in length. In another embodiment, the sequence is selected from the group consisting of residues 9 to 59 of SEC NO. OF IDENTIFICATION: 2 and residues from 9 to 59 of SEC NO. IDENTIFICATION: 4. In a further embodiment, the protein consists of 51-557 contiguous amino acid residues of SEC NO. IDENTIFICATION: 10. In a further embodiment, the protein additionally comprises an affinity aggregate. Suitable affinity aggregates include binding protein with maltose, polyhistidine, and Glu-Tyr-Met-Pro-Met-Glu (SEQ ID NO: 18).

In a second aspect, the invention provides an expression vector comprising the following operable linked elements: (a) a transcription promoter; (b) a segment of DNA encoding a protein as described above; and (c) a transcription terminator. In one embodiment, the expression vector further comprises a secretory signal sequence linked in operable form to the DNA segment.

In a third aspect, the invention provides a cultured cell that contains an expression vector as described above, wherein the cell expresses the DNA segment.

In a fourth aspect of the invention a method is provided for the preparation of protein with proteinase inhibitory activity comprising the cultivation of a cell as described above under conditions through which the DNA segment is expressed, and the recovery of the protein encoded by the DNA segment.

In a fifth aspect of the invention, an antibody is provided that specifically binds to a protein as shown in SEQ. NO. IDENTIFICATION: 2 or in SEC NO. IDENTIFICATION: 4.

These and other aspects of the invention will become apparent with reference to the following detailed description and the accompanying drawing.

BRIEF DESCRIPTION OF THE DRAWING Figure 1 shows an amino acid sequence alignment of a representative polypeptide of the present invention (SEQ.ID.NO.:2), designated "ZKUN5", with the Kunitz domain sequence of human collagen alpha 3 type VI (SEC NO. IDENTIFICATION: 8), designated as "1KNT".

DETAILED DESCRIPTION OF THE INVENTION Before describing the invention in detail, it will be helpful for your understanding to define the following terms: The term "affinity aggregate" is used herein to denote a polypeptide segment that can bind to a second polypeptide to provide purification of the second polypeptide or provide sites for the attachment of the second polypeptide to a substrate. In principle, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity aggregate. Affinity aggregates include a poly-histidine tract, protein A (Nilsson et al., EMBO J. 4: 1015, 1985; Nilsson et al., Methods Enzimol. 198: 3, 1991), glutathione S transferase (Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity aggregate (Grussenmeyer et al., Proc. Na ti.Acid. Sci. USA 82: 7952-4, 1985) (SEQ ID NO: 18), substance P, Flag ™ peptide (Hopp et al., Biotechnology 6: 1204-1210, 1988), streptavidin binding peptide, maltose binding protein (Guan et al., Gene j57: 21-30, 1987), cellulose binding protein, thioredoxin, ubiquitin, T7 polymerase, or other antigenic epitope or binding domain. See generally, Ford et al., Protein Expression and Purification 2: 95-107, 1991. Affinity aggregates of DNA conditioners and other reagents are available from commercial distributors (eg, Pharmacia Biotech, Piscataway, NJ; New England Biolabs, Beverly, MA; Eastman Kodak, New Haven, CT).

The term "allelic variant" is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal position. Allelic variation arises naturally through mutation, and can result in a phenotypic polymorphism within the populations. Mutations of genes can be silent (without change in the encoded polypeptide) or can encode polypeptides having an altered amino acid sequence. The term allelic is also used here to denote a protein encoded by an allelic variant of a gene.

The terms "amino terminus" and "carboxyl terminus" are used herein to denote positions in the polypeptides. Wherever the context permits, these terms are used with reference to a particular sequence or portion of a polypeptide to denote a proximity or relative position. For example, a certain carboxyl terminal with position in a sequence with respect to a reference sequence in a polypeptide is located near the carboxyl terminus of the reference sequence, but is not necessary at the carboxyl terminus of the complete polypeptide.

A "complement" of a polynucleotide molecule is a polynucleotide molecule having a complementary base sequence and a reverse orientation as compared to a reference sequence. For example, the 5 'sequence ATGCACGGG 3' is complementary with 5 'CCCGTGCAT 3'.

The term "degenerate nucleotide sequence" denotes a nucleotide sequence that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide). The degenerate codons contain different trios of nucleotides, but they encode the same amino acid residue (ie, the trios GAU and GAC encode each Asp).

A "DNA segment" is a portion of a larger DNA molecule that has specified attributes. For example, a DNA segment encoding a specified polypeptide is a portion of a larger DNA molecule, such as a plasmid or a plasmid fragment, which, when read from the 5 'to 3A direction encodes the amino acid sequence of the specified polypeptide.

The term "expression vector" is used to denote a linear or circular DNA molecule, comprising a segment encoding a polypeptide of interest linked in operable form to additional segments that it provides for transcription. Such additional segments include promoter and terminator sequences, and may also include one or more duplication sources, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.

The term "isolated", when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic environment and is therefore free of other foreign or unwanted coding sequences, and is in a form suitable for use in systems of protein production by genetic engineering. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. The isolated DNA molecules of the present invention are free of other genes with which they are normally associated, but may include 5 'and 3' non-translated regions of natural occurrence such as promoters and terminators. The identification of associated regions will be evident to someone with normal experience in the art (see, for example, Dynan and Tijan, Na ture 316: 114-18, 1985).

An "isolated" polypeptide or protein is a polypeptide or a protein that is in a condition different from its native environment, such as separated from blood and animal tissue. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, ie, more than 95% pure, more preferably more than 99% pure. When used within this context, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as coiro dimers or alternatively glycosylated or derivative forms.

The term 'linked in operable form', when referring to DNA segments indicates that the segments are arranged in such a way that they work together for their intended purposes, for example, the transcription starts at the promoter and proceeds through the segment of coding or finisher.

The term "Ortholog" denotes a polypeptide or protein obtained from a species that is the functional counterpart of a polypeptide or protein of different species.Speed differences between orthologs are the result of speciation.

A 'polynucleotide' is a single-stranded polymer or double deoxyribonucleotide or ribonucleotide bases read from the 5 'to the 3' end.The polynucleotides include RNA and ADK, and can be isolated from their natural sources, sitetized in vi tro, or prepared from a combination of natural or synthetic molecules The sizes of the polynucleotides are expressed as base pairs (abbreviated as 'bp'), nucleotides ('nt'), or kilobases ('kb'). When the context permits, the last two terms describe polynucleotides that are single-stranded or double-stranded. When these terms are applied to double-stranded molecules they are used to denote the total length and will be understood to be equivalent to the term "base pairs." Those skilled in the art will recognize that two strands of a double-stranded polynucleotide may differ slightly. in length and that their ends may be staggered as a result of enzymatic cleavage, therefore all nucleotides in a double-stranded polynucleotide molecule may not be paired, such odd ends generally not exceeding 20 nt in length.

A "polypeptide" is a polymer of amino acid residues linked by peptide bonds, whether produced naturally or synthetically, polypeptides of less than 10 amino acid residues are commonly referred to as 'peptides'.

The term "promoter" is used herein for its recognized meaning in the art to denote a portion of a gene that contains DNA sequences that provides for the binding of RNA polymerase and the initiation of transcription. Promoter sequences are commonly, but not always, found in the regions of non-coding 5 'genes.

A "protein" is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents can be added to a protein by means of the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined here in terms of their major amino acid structures; substituyent.es such as carbohydrate groups are generally not specified, but nevertheless may be present.

The term "secretory signal sequence" denotes a DNA sequence encoding a polypeptide (a "secretory peptide") which, as components of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which It is synthesized. The larger polypeptide is commonly divided to remove the secretory peptide during transit through the secretory pathway.

The term "splice variant" is used here to denote alternative forms of RNA transcribed from a gene. The variation of splicing arises naturally through the alternative use of splice sites in a transcribed RNA molecule, less commonly between transcribed RNA molecules, and can result in several transcribed mRNAs or the same gene. The splice variants can encode polypeptides having an altered amino acid sequence. The term "splice variant" is also used herein to denote a protein encoded by a splicing variant of a mRNA transcribed from a gene.

The molecular weights and lengths of the polymers determined by imprecise analytical methods (for example, gel electrophoresis) should be understood as approximate values. When a value of that type is expressed as "about" X or "about" X, the set value of X will be understood to have an accuracy of ± 10%.

The present invention provides, in part, serine proteinases comprising a Kunitz domain. This Kunitz domain, which includes its sequence variants and proteins that contain it, is referred to here as "zkun5". The polypeptide sequence of zkun5 shown in SEQ. DO NOT. IDENTIFICATION: 2 comprises this Kunitz domain, which is delimited at the amino and carboxyl terminals by means of cysteine residues at positions 9 and 59 respectively.

Zkun5 has 50% residue identity with the kunitz domain in human collagen alpha 3 type VI (shown in SEQ ID NO: 8). The structure of this last domain has been solved by X-ray crystallography and by NMR (Arnoux et al., J. Mol. Biol. 246: 609-617, 1995, Sorensen et al., Biochemistry 36: 10439-10450, 1997). An alignment of the zkun5 and the Kunitz domain of collagen (see Figure 1) can be combined with a homology model of zkun5 based on the X-ray structure to predict the function of certain residues in the zkun5. With reference to the SEC. DO NOT. IDENTIFICATION: 2, disulfide bonds are predicted to be formed by paired cysteine residues Cys9-Cys59; Cysld-Cys42; and Cys34-Cys55. The protease binding loop (P3-P4 ') is expected to comprise residues 17-23 of the SEC. DO NOT. IDENTIFICATION: 2 (Asn-Cys-Gly-Glu-Tyr-Val-Val), the residue being Pl Glyl9 and the residue being Pl 'Glu20. Zkun5 additionally comprises a potential glycosylation site at position Asn43 of SEC. DO NOT. IDENTIFICATION: 2.

It is not known that the kunitz domain of human alpha 3 type VI collagen has antiproteinase activity. Suspected reasons for this include unfavorable steric hindrances with trypsin that involve the Aspl5 residue of the collagen (corresponding to Glu20 in zkun5). From these data it is predicted that a Glu20Ala mutant of zkun5 may show increased antiproteinase activity. Therefore, the present invention contemplates zkun5 proteins wherein residue 20 of SEQ. DO NOT. ID: 2 is replaced with Wing as shown in SEC. DO NOT. IDENTIFICATION: 4.

Additional amino acid substitutions can be made in the zkun5 sequence as long as the conserved cysteine residues are retained and the higher order structure is not disturbed. It is preferred to make substitutions in the Kunitz domain of zkun5 with reference to the sequences of other Kunitz domains. The SEC. DO NOT. IDENTIFICATION: 5 is a generalized Kunitz domain sequence that shows allowable amino acid substitutions based on such alignment. The sequence of residue 51 shown in SEQ. DO NOT. IDENTIFICATION: 5 is in accordance with the pattern: C-X (8) -C-X (15) -C-X (7) -C-X (12) -C-X (3) -C where C denotes cysteine, X is any naturally occurring amino acid residue, subject to the limitations set forth in the Sequence Listing annexed to the SEC. DO NOT. IDENTIFICATION: 5; and the numbers indicate the number of such variable residues. The second cysteine residue is in the P2 position.

In the present invention up to 10% of amino acid residues in the Kunitz domain of zkun5 (residues 9 to 59 of SEQ ID NO: 2) can be replaced with other amino acid residues, subject to the limitation of cue sequence substituted substitute is one of the sequences represented in the SEC. DO NOT. IDENTIFICATION: 5. Therefore, the present invention provides a family of proteases comprising a sequence of amino acid residues as shown in SEQ. DO NOT. IDENTIFICATION: 5, where the sequence is at least 90% identical to residues 9 to 59 of the SEC. DO NOT. OF IDENTIFICATION: 2. It is preferred that the proteins of the present invention comprise a sequence such that it is at least 95% identical to residues 9 to 50 of SEQ. DO NOT. IDENTIFICATION: 2.

The percent sequence identity is determined by conventional methods. See for example, Altschul et al., Bull. Ma th. Bio. 48: 603-616, 1986, and Henikoff and Henikoff, Proc. Nati Acad. Sci. USA 9: 10915-10919, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment markers using a space opening penalty of 10, a space extension penalty of 1, and the "BLOSUM62" marking matrix. from Henikoff and Henikoff (mentioned above) as shown in Table 2 (amino acids are indicated by standard one-letter codes). The identity percent is then calculated as: Total number of identical pairs x 100 [length of the largest sequence plus the number of spaces entered within the larger sequence in order to align the two sequences] Table 1 RNACQEGHILKMFPSTWYVA 4 R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0 2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 L -1 -2 - 3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 - 3 -2 1 2 -1 5 F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1 - 1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 - 3 -2 11 And -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4 The level of identity between amino acid sequences can be determined using the search algorithm of similarity "FASTA" by Pearson and Lipman (Proc. Nati, Acad. Sci. USA 85: 2444, 1988) and Pearson. { Meth. Enzymol. 183: 63, 1990). Briefly, FASTA first characterizes the similarity of the sequence by identifying regions shared by the sequence in question (eg, SEC.NO IDENTIFICATION: 2) and a test sequence that has the highest density of identities (if the variable ktup is 1) or pairs of identities (if ktup = 2), without considering conservative amino acid substitutions, insertions, or deletions. The ten regions with the highest density of identities are then highlighted by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "arranged" to include only those residues that contribute to the highest score. . If there are several regions with markings greater than the "cut" value (calculated by means of a predetermined formula based on the length of the sequence and the value of ktup), then the initial regions arranged are examined to determine if the regions can join to form an approximate alignment with the spaces. Finally, the regions with the highest labeling of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48: 444, 1970; Sellers, SIAM J. Appl. Ma th. 26: 787, 1974), which allows the insertion and deletion of amino acids. Illustrative parameters for the analysis of FASTA sin: ktup = l, space opening penalty = 10, space extension penalty = 1, and substitution matrix = BLOSUM62. These parameters can be entered into the FASTA program by modifying the file of the dial matrix ("SMATRIX"), as explained in Appendix 2 of Pearson, 1990 (previously mentioned).

FASTA can also be used to determine the sequence identity of the nucleic acid molecules using a ratio as described above. For comparisons of nucleotide sequences, the value of ktup can vary between one and six, preferably four to six.

The proteins of the present invention may also comprise amino acid residues of non-natural occurrence. Amino acids of unnatural occurrence include, without limitation, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycyl, allo-threonine, methyltreonine, hydroxyethylcystira, hydroxyethylhomocysteine, nitroglutamine , homoglutamine, pipecolic acid, thiazolidine carboxylic acid, idroproline, 3-and 4-methylproline, 3, 3-dimethylproline, ter-leucine, norvalir.a, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine . Various methods are known in the art for the incorporation of amino acid residues of non-natural occurrence within the proteins. For example, an in vi tro system comprising an extract of E. coli S30 and commercially available enzymes and other reagents can be used, where nonsense mutations are suppressed using chemically aminociclated tRNA suppressors. See for example, Robertson et al., J. Am. Chem. Soc. 113: 2722, 1991; Ellman et al., Methods Enzymol. 202: 301, 1991; Chung et al., Science 259: 806-9, 1993; and Chung et al., Proc. Na ti. Acad. Sci. USA 9 ^: 10145-9, 1993). In a second method, the translation is carried out in Xenopus oocysts by microinjection of mutated mRNA and chemically aminociclated tRNA suppressors.

(Turcatti et al., J. Biol. Chem. 271: 1999-1-8, 1996).

In a third method, E. coli cells are cultured in the absence of a natural amino acid that will be replaced (e.g., phenylalanine) and in the presence of the non-naturally occurring amino acid (e.g., 2-azaphenylalanine, 3). -azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). See Koide et al., Biochem. 33: 7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by chemical modification in vitro. Chemical modification may be combined with site-directed mutagenesis to further extend the range of substitutions (Wynn and Richards, Prote n Sci. 2: 395-403, 1993).

Additional polypeptides can be attached to the amino and / or carboxyl terminal of the Kunitz domain of zkun5 (residues 9-59 of SEQ ID NO: 2) or a Kunitz domain derivative of zkun5 as described above. In one modality, the extensions are those shown in the SEC. DO NOT. IDENTIFICATION: 10, where the Kunitz domain of zkun5 is located in residues 504-554. Particularly preferred proteins in this regard include residues 1-62 of SEQ ID NO.:2 or SEC. ID NO.:4. The amino and carboxyl spreads of the Kunitz domain of zkun 5 will be selected so that the proteinase inhibitory activity of the protein is not destroyed or masked, for example by hiding the Kunitz domain within the interior of the protein. There is a consequent preference for short extensions, typically residues 10-15 in length, preferably not exceeding 8 residues in length. There is a considerable latitude in the permissible sequence of these extensions, although it is preferred to avoid the addition of cysteine residues in close proximity to the ,. «« Same domain Kunitz. For example, a zkun5 protein may comprise residues 9-59 of SEC. DO NOT. IDENTIFICATION: 2 or the SEC. DO NOT. IDENTIFICATION: 4 with amino- and carboxyl- terminal dipeptides, wherein the individual amino acid residues of the dipeptides are any amino acid residue except cysteine.

Other amino and carboxyl terminal extensions that can be included in the proteins of the present invention include, for example, an amino terminal methionine residue, a small binding peptide of up to about 20 -5 residues or an affinity aggregate as described earlier. A protein comprising such an extension may comprise a polypeptide linker and / or a proteolytic cleavage site between the zkun5 portion and the affinity aggregate. Preferred cleavage sites include thrombin cleavage sites and factor Xa cleavage sites. For example, the zkun5 protein shown in SEC. DO NOT. IDENTIFICATION: 2 can be expressed as a fusion comprising, from an amino terminus to a carboxyl terminus: a maltose-binding protein-polyhistidine-dividing site (Leu-Val-Pro-Arg; SEQ. IDENTIFICATION: 1 !.) -SEC. DO NOT. IDENTIFICATION: 2. Linker peptides and affinity aggregates provide additional functions, such as binding to substrates, antibodies, binding proteins, and the like, and facilitate the purification, detection, and delivery of zkun5 proteins. In another example, a Kunitz domain of zkun5 can be expressed as a secreted protein comprising a transmembrane carboxyl terminal receptor domain, which allows the Kunitz domain to be displayed on the surface of the cell. To encompass the bilayer lipid of the cell membrane, a minimum of 20 amino acids is required in the transmembrane domain; these would predominantly be hydrophobic amino acids. The Kunitz domain can be separated from the transmembrane domain by a spacer polypeptide, and can be contained in an extended polypeptide comprising a polypeptide of the carboxyl terminal polypeptide of the amino terminal Kunitz domain spacer polypeptide. Many transmembrane receptor and polynucleotide domains encoding them are known in the art. The spacer polypeptide will generally be at least 50 amino acid residues in length, up to 200-300 or more residues. The amino terminal polypeptide may be up to 300 or more residues in length.

The present invention provides additional polynucleotide molecules, including DNA and RNA molecules, which encode zkun5 proteins. The polynucleotides of the present invention include the strand in the 5 'direction; the strand in the 3 'direction; and the DNA as a double strand, both having the strand of sense and antisense tempered by their respective hydrogen bonds. Representative sequences of DNA encoding zkun5 proteins are presented in SEQ. ID NO .: 1, SEC. ID. NO .: 3, and SEC. ID NO .: 9. DNA sequences encoding other zkun5 proteins can be easily generated by those with normal experience in the art based on the genetic code. The counterparts of RNA sequences can be generated by substitution of U by T.

Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible between these polynucleotide molecules. The SEC. DO NOT. IDENTIFICATION: 6 is a degenerate DNA sequence comprising all the DNAs encoding the zkun5 polypeptide of SEC. DO NOT. IDENTIFICATION: 2. The SEC. DO NOT. IDENTIFICATION: 7 is a degenerate DNA sequence comprising all the DNAs encoding the zkun5 polypeptide of the SEC. DO NOT. IDENTIFICATION: 4. Those skilled in the art will recognize that the degenerate sequences of the SEC. DO NOT. IDENTIFICATION: 6 and SEC. DO NOT. IDENTIFICATION: 7 also provide RNA sequences that encode the SEC. DO NOT. IDENTIFICATION: 2 and the SEC. DO NOT. IDENTIFICATION: 4, respectively, by the substitution of U by T. Thus, polynucleotides encoding zkun5 polypeptide comprising nucleotide 1 to nucleotide 186 of SEQ. DO NOT. IDENTIFICATION: 6, nucleotide 1 to nucleotide 186 of SEC. DO NOT. IDENTIFICATION: 7, and their respective RNA equivalents are contemplated by the present invention. Table 2 presents the one-letter codes used in the SEC. DO NOT. IDENTIFICATION: 6 and 7 to denote degenerate nucleotide positions. The "resolutions" are the nucleotides denoted by a code letter. The "complement" indicates the code for the complementary nucleotide (s). For example, the code Y denotes C or T, and its complement R denotes A or G, being A complementary to T, and G complementary to C.

Table 2 Nucleotide Resolution Nucleotide Complement A A T T C C G G G G C C T T A A RA | GYC | TYC | TRA | GMA | CKG | TKG | TMA | CSC | GSC | G w A | TWA | TH AICIT DC | G | TB CIGIT VA | C | GVA | C | GB CIGIT DA | G | THA | C | TN AICIGIT N AICIGIT The degenerate codons used in SEC. DO NOT. IDENTIFICATION: 6 and 7, which covers all possible codons for a given amino acid, are shown in Table 3.

Table 3 Single Codon Code Amino Acid Letter Degenerate Codons _______ __ Cys C Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACÁ ACC ACG ACT ACN Pro P CCA CCC CCG CCT CCN Wing A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AAR Met M ATG ATG He I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTN Val V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr and TAC TAT TAY Trp W TGG TGG Ter TAA TAG TGA TRR Asn | Asp B RAY GluIGln Z SAR Any X NNN Someone with normal experience in the art will appreciate that some ambiguity is introduced in the determination of a degenerate codon, representative of all possible codons that encode each amino acid. For example, the degenerate codon for serine (WSN) can, in some circumstances, encode arginine (AGR) and the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY). A similar relationship exists between codons that encode phenylalanine and leucine. therefore, some polynucleotides comprised by the degenerate sequence can encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequences shown in SEQ. DO NOT. IDENTIFICATION: 2 and 4. Variant sequences can be easily tested for functionality as described herein.

Someone with ordinary knowledge in the art will also appreciate that different species may exhibit a preferential codon usage, that is, a deviation from the use of the codon in the genome of a species. See, in general, Grantham et al., Nuc. Acids Res. 8: 1839-912, 1980; Haas et al., Curr. Biol. 6: 315-24, 1996; Wain-Hobson et al., Gene 1_3: 355-64, 1981; Grosjean and Fiers, Gene 18: 199-209, 1982; Holm, Nuc. Acids Res. 14: 3075-87, 1986; and Ikemura, J. Mol. Biol. 158: 573-97, 1982. Preferred codons for a particular species can be introduced into the polynucleotides of the present invention by means of a variety of methods known in the art, thus increasing translation efficiency in a particular type of cell or species. The degenerate codon sequences represented in SEQ. DO NOT. IDENTIFICATION: 6 and 7 serve as a standard for optimizing the expression of polynucleotides in various cell types and species commonly used in the art and described herein. The sequences containing preferred codons can be tested and optimized for expression in several host cell species, and tested for functionality as described herein.

It is preferred that zku: 5 polynucleotides hybridize to similar regions of SEQ. DO NOT. IDENTIFICATION:!, Or a sequence complementary to these, under strict conditions. In general, stringent conditions are selected to be 5 ° C lower than the thermal melting point (Tm) for the specified sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched sample. Typically strict conditions are those in which the salt concentration is up to 0.03 M at a pH of 7 and the temperature is at least about 60 ° C.

As noted above, the zkun5 polynucleotides provided by the present invention include DNA and RNA. Methods for the preparation of DNA and RNA are well known in the art. In general, RNA is separated from a tissue or cell that produces large amounts of zkun5 RNA. Such tissues and cells are identified by the Northern Blotting technique (Thomas, Proc. Nati, Acad. Sci. USA 77: 5201, 1980), and include spinal cord, trachea, heart, colon, small intestine, and stomach. Total RNA can be prepared using guanidine-HCl extraction followed by separation by centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry _18: 52-94, 1979). Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Nati, Acad. Sci. USA < 69: 1408-12, 19721. Complementary DNA (cDNA) is prepared from poly (A) + RNA using known methods Alternatively, genomic DNA can be separated, then polynucleotides encoding zkun5 polypeptides are identified and separated for example, by hybridization or by PCR.

A full-length clone encoding zkun5 can be obtained by conventional cloning procedures. Preferred are complementary DNA clones (cDNA), although for some applications (e.g., expression in transgenic animals) it will be preferable to use a genomic clone, or modify a cDNA clone to include at least one genomic intron. Methods for the preparation of cDNA and genomic clones are well known and for the level of someone skilled in the art, and include the use of the sequence described herein, or its parts, for the sampling or preparation of a library. Expression libraries can be sampled with antibodies to zkun5, receptor fragments, or other specific binding partners.

The polynucleotides of the present invention can also be synthesized using automated equipment ("gene machines"). Methods of gene synthesis are well known in the art. See, for example, Glick and Pasternak, Molecular Biotechnology, Principies & Applications of Recombinant DNA (Molecular Biotechnology, Principles and Applications of Recombinant DNA), ASM Press, Washington, D. C, 1994; Itakura and collaborators, Annu. Rev. Biochem. 53: 323-356, 1984; and Cumie et al., Proc. Nati Acad. Sci. USA 87: 633-637, 1990.

The polynucleotide sequences of zkun5 described herein can be used to isolate the polynucleotides counterparts of other species (orthologs). These orthologous polynucleotides can be used, inter alia, to prepare the respective orthologous proteins. These other species include, but are not limited to, mammals, birds, amphibians, reptiles, fish, insects and other vertebrate and invertebrate species. Of particular interest are the zkun5 abd polypeptide polynucleotides of other mammalian species, including mice, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. The orthologs of human zkun5 can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses zkun5 as described herein. Suitable mRNA sources can be identified by Northern blot sampling with samples designed from the sequences described here. A mRNA library of a positive tissue or cell line is then prepared. Then a cDNA that encodes ».a? -A" * .'- zkun5 can be isolated by a variety of methods, such as sampling with a complete or partial human cDNA or with one or more sets of degenerated samples in the sequences depicted. A cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent No. 4,683,202), using designed primers of the human zkun5 sequence described herein. In a further method, the cDNA library can be used to transform or txansfect host cells, and expressions of the cDNA of interest can be detected with an antibody to the zkun5 polypeptide. Similar techniques can also be applied in the separation of genomic clones.

Those skilled in the art will recognize that the sequence represented in the SEC. DO NOT. IDENTIFICATION:! represents a single allele of human zkun5 and that natural variation is expected to occur, including allelic variation and alternative splicing. The allelic variants of this sequence can be cloned by sampling cDNA or genomic libraries from different individuals according to standard procedures. The allelic variants of the DNA sequence shown in SEQ. DO NOT. FROM IDENTIFICATION: 1 including silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins that are allelic variants of SEC. DO NOT. OF IDENTIFICATION: 2. The cDNAs generated from alternatively spliced mRNAs, which retain the inhibitory activity of zkun5 proteinase are included within the scope of the present invention, co-polypeptides encoded by such cDNAs and mRNAs. The allelic variants and splice variants of these sequences can be cloned by sampling cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art.

Zkun 5 proteins, including wild type zkun5 variants, are tested for activity in protease inhibition assays, a variety of which are known in the art. Preferred assays include those that measure inhibition of trypsin, chymotrypsin, plasmin, cathepsin G, and human leukocyte elastase. See, for example, Petersen et al., Eur. J. Biochem. 235: 310-316, 1996. In a typical procedure, the inhibitory activity of a tested compound is measured by incubation of the test compound with proteinase, then adding an appropriate substrate, typically a chromogenic peptide substrate. See, for example, Norris et al. (Biol. Chem. Hoppe-Seyler 371: 37-42, 1990). Briefly, several concentrations of the inhibitor are incubated in the presence of trypsin, plasmin, and plasma kelikrein in a low-salt buffer solution at a pH of 7.4, 25 C. After 30 minutes, the residual enzyme activity is measured by the addition of a chromogenic substrate (eg, S2251 (D-Val-Leu- Lys-Nan) or S2302 (D-Pro-Phe-Arg-Nan), available from Kabi, Stockholm, Sweden) and a 30 minute incubation. The inhibition of the enzymatic activity is indicated by a decrease in the absorbance at 405 nm of Em fluorescence at 460 nm. From these results, the apparent inhibition constant Ki is calculated. Inhibition of coagulation factors (eg factor Vlla, factor Xa) can be measured using chromogenic substrates or in conventional coagulation assays (eg, human plasma coagulation time); Dennis et al., Cited above).

Zkun5 proteins can be tested in animal disease models, particularly tumor models, fibrinolysis models, and models of hemostasis imbalance. Appropriate models are known in art. For example, inhibition of tumor metastasis can be evaluated in mice within which cancer cells or tumor tissues have been introduced by implantation or injection (eg, Brown, Advan, Enzyme Regul. 35: 293-301, 1995; Conway et al., Clin. Exp. Metastasis 14_: 115-124, 1996). The effects on fibrinolysis can be measured in a rat model in which the radiolabelled batroxibin and fibrinogen enzyme are administered to test animals. The inhibition of fibrinogen activation by a test compound is seen as a reduction in the level of circulation of the label when compared in animals that do not receive the test compound. See, Lenfors and Gustafsson, Semin. Thromb. Hemost. 22: 335-342, 1996. Zkun5 proteins can be delivered to test animals by injection or infusion, or they can be produced in vivo by, for example, viral or defenseless DNA systems or by transgenic expression.

Example viral delivery systems include adenovirus, herpesvirus, vaccinia virus and adeno-associated virus (AAV). The adnovirus, a double-stranded DNA virus, is usually the smallest gene transfer vector studied for the delivery of heterologous nucleic acid (for a review, see Becker et al., Meth. Cell. Biol. 43: 161-189, 1994; and Douglas and Curiel, Science &Medicine: 44'53, 1997). By deleting portions of the adenovirus genome, large inserts (up to 7 kb) of heterologous DNA can be accommodated. These inserts can be incorporated into the viral DNA by direct binding or by homologous recombination with a co-transfected plasmid. In an exemplary system, the essential gene El is deleted from the viral vector, and the virus will not be duplicated unless the El gene is provided by the host cell (eg, the human cell line 292). When administered intravenously in intact animals, the adenovirus first attacks the liver. If the adenoviral delivery system has a deletion of the El gene, the virus can not be duplicated in the host cell. However, the host tissue (e.g., the liver) will express and process (and, if there is a signal sequence, secrete) the heterologous protein. The secreted proteins will enter the circulation in the highly vascularized liver, and the effects on the infected animal can be determined.

An alternative method of gene delivery involves the removal of cells from the body and the introduction of a vector into the cells as a defenseless DNA plasmid. The transformed cells are then re-implanted in the body. Defense-free DNA vectors are introduced into host cells by methods known in the art, including transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene trigger, or use of a DNA vector transporter. See Wu et al., J. Biol. Chem. 263: 14621-14624, 1988; Wu et al., J. Biol. Chem. 267: 963-967, 1992; and Johnston and ~ sA Tang, Meth. Cell. Biol. 43: 353-365, 1994.

Transgenic mice, which by engineering express a zkun5 gene and mice that exhibit a complete absence of zkun5 gene function, referred to as "knockout mice" (Snouwaert et al., Science 257: 1083, 1992), may also be generated (Lowell et al., Na ture 366: 740-742, 1993). These mice are used to study the zkun5 gene and the encoded protein in a system in vivo. Transgenic mice are particularly useful for investigating the role of zkun5 proteins in early development because they allow the identification of developmental abnormalities or blockages that result from the over or under-expression of a specific factor.

The zkun5 polypeptides of the present invention, including full-length polypeptides, biologically active fragments, and fusion polypeptides, can be produced in genetically engineered host cells according to conventional techniques. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungoid cells, and major eukaryotic cells cultured. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are described by Sambrook et al., Molecular Cloning: A Laboratory Manual (Molecular Cloning: A Laboratory Manual), 2a. edition, Cold Spring Harbor Laboratory Express, Cold Spring Harbor, NY, 1989, and Ausubel et al., editors, Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987.

In general, a DNA sequence encoding a zkun5 polypeptide is opey linked to other genetic elements required for its expression, which generally includes a transcription promoter and a terminator, within an expression vector. The vector will also commonly contain one or more selectable markers and one or more duplication origins, although those skilled in the art will recognize that within certain systems the selectable markers can be provided in separate vectors, and the duplication of the exogenous DNA can be provided by integration into a host cell genome. The selection of promoters, finalizers, selectable markers, vectors and other elements is a matter of routine design within the level of someone with experience in the art. Many such elements are described in the literature and are available through commercial distributors.

To direct a zkun5 polypeptide within the secretory pathway of a host cell, a secretory serial sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence may be that of zkun5, or it may be derived from another secreted protein (e.g., t-PA; see U.S. Patent No. 5,641,655) or synthesized again. The secretory signal sequence is opey linked to the zkun5 DNA sequence, ie, the two sequences are linked in the correct reading frame and are positioned to direct the new synthesized polypeptide into the secretory pathway of the host cell . Secretory signal sequences are commonly placed 5 'to the DNA sequence encoding the polypeptide of interest, although some sequence signals can be placed anywhere in the DNA sequence of interest (see, eg, Welch et al. U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830).

Cultured mammalian cells are suitable hosts for use in the invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14: 725, 1978; Corsaro and Pearson, Soma tic Cell Genetics 7: 603, 1981; Graham and Van der Eb , Virology 52: 456, 1973), electroporation (Neumann et al., EMBO J. 1: 841-845, 1982), DEAE transfection mediated by dextran (Ausubel et al., Cited above), and liposome-mediated transfection (Hawley-Nelson and collaborators, Focus 1_5: 73, 1993; Ciccarone et al., Focus 15:80, 1993). The production of recombinant polypeptides in cultured cells of mammals are described, for example, by Levinson et al., In US Patent no. 4,713,339; Hagen et al., US Patent No. 4,784,950; Palmiter et al., US Patent No. 4,579,821; and Ringold, US Patent No. 4,656,134. Suitable mammalian cultured cells include cell lines COS-1 (ATCC No. CRL 1650), COSA (ATCC No. CRL 1651) BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen Virol. 36: 59-72, 1977) and Chinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61). Additional suitable cells are known in the art and are available from public storages such as the American Type Culture Collection, Rockville, Maryland. In general, strong transcription promoters, such as SV-40 or cytomegalovirus promoters, are preferred. See, for example, U.S. Patent No. 4,956,288. Other suitable promoters include those of the metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter. Expression vectors for use in mammalian cells include pZP-1 and pZP-9, which have been deposited at the American Type Culture Collection, Rockville, MD, United States of America based on accession numbers 98669 and 98668, respectively.

The selection of the drug is generally used to select for cultured cells of mammals into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants." Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as "stable transfectants." A preferred selectable marker in a gene encoding resistance to antibiotic neomycin. The selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like. Selection systems can also be used to increase the level of expression of the gene of interest, a process referred to as 'amplification.' Amplification is carried out by culturing transfectants in the presence of a low level of selective agent and the subsequent increase in the amount of selective agent to select the cells that produce high levels of the products of the introduced genes A preferred selectable amplifiable marker is dihydrofolate: or reductase, which confers resistance to methotrexate Other resistant genes can also be used to drugs (for example, puromycin acetyltransferase, resistant to hygromycin, resistant to multiple drugs).

Other higher eukaryotic cells can also be used as hosts, including insect cells, plant cells and bird cells. The use of Agrobacterium um rhizogenes as a vector for the expression of genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 1: 1: 47-58, 1987. The transformation of insect cells and the production there of foreign polypeptides is described by Guarino et al., US Patent No. 5,162,222 and the OMPI publication WO 94/06463.

Insect cells can be infected with recombinant baculoviruses, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV) using methods commonly known in the art. See, King and Posee, The Baculovirus Expression System: A Laboratory Guide (The Baculovirus Expression System: A Laboratory Guide), London, Chapman & Hall; O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual (Expression Vectors of Baculovirus: A Laboratory Manual), New York, Oxford Press University, 1994; and Richardson, Editor, Baculoviros Expression Protocols. Methods in Molecular Biology, Humana press, Totowa, NJ, 1995. Recombinant baculoviruses can also be produced by the use of a transposon-based system described by Luckow et al. [J. Virol. . íl_: 4566-4579, 1993). This system, which uses transfer vectors, is commercially available in the form of equipment (Bac-to-Bac ™ equipment, Life Technologies, Rockville, MD). The transfer vector (eg, pFastBacl, Life Technologies) contains a Tn7 transposon to move the DNA encoding the protein of interest within a baculovirus genome maintained in E. Coli as a large plasmid called a "bacmid." See Hill-Perkins and Possee, J. Gen. Virol. 71: 971-976, 1990, Bonning et al., J. Gen. Virol. 75: 1551-1556, 1994, and Chazenbalk and Rapoport, J. Biol. Chem. 270 : 1543.1549, 1995.

The fungoid cells, including yeast cells, can also be used in the present invention. Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica. Methods for the transformation of S. cerevisiae cells with exogenous DNA and the production of recombinant polypeptides therefrom are described, for example, by Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al., U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al., US Patent No. 5,037,743; and Murray et al., US Patent No. 4,845,075. Transformed cells are selected by the phenotype determined by the selectable marker, commonly drug resistant or the ability to grow in the absence of a particular nutrient (e.g., leucine). A preferred vector system for use in Saccharomyces cerevisi ae is the POT1 vector system described by Kawasaki et al. (U.S. Patent No. 4,931,373) which allows transformed cells to be selected by growth in a medium containing glucose. Suitable promoters and terminators for use in yeast include those of the glycolytic enzyme genes (see, for example, Kawasaki, US Patent No. 4,599,311, Kingsman et al., US Patent No. 4,615,974, and Bitter, US Patent No. 4,977,092) and genes alcohol dehydrogenase. See also Patents US Pat. Nos. 4,990,446, 5,063,154, 5,139,936 and 4,661,454 Transformation systems are known in the art for other yeasts including Hansenula polymorph, Schizosaccaromyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis. , Pichia pastoris, Pichia methanolica, Pichia guillermondii, and Candida maltose See, for example, Gleeson et al., J. Gen. Microbiol., 132: 3459-3465, 1986 and Cregg, US Patent No. 4,882,279 Aspergillus cells can be used for according to the methods of McKnight et al., U.S. Patent No. 4,935,349 The methods for the transformation of Acremonium chrysogenum are described by Su ino et al., US Patent No. 5,162,228 The methods for the transformation of Neurospora are described by Lambowitz, Patent No. 4,486,533 The use of Pichia methanolica as a host for the production of recombinant proteins is described ibe in U.S. Patents Nos. 5,716,808, 5,736,383, 5,854,039, and 5,888,768; and in the OMPI publications WO 97/17450 and WO 97/17451.

Prokaryotic host cells, including strains of the bacterium Escherichia coli, Bacillus and another genus are also useful host cells in the present invention. The techniques for the transformation of these hosts and the expression of external DNA sequences cloned there are well known in the art (see, for example, Sambrook, et al., Cited above). When a zkun5 polypeptide is expressed in a bacterium such as _. Coli, the polypeptide can be retained in the cytoplasm, typically as insoluble granules, or it can be directed to the periplasmic space by a sequence of bacterial secretion. In the above case, the cells undergo a break, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can then be remultiplicated and dimerized by diluting the denaturant, such as, by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. In the latter case, the polypeptide can be recovered from the periplasmic space in a soluble and functional form by means of the rupture of cells (for example, by sonication or osmotic shock) to release the content of the periplasmic space and recover the protein, thus obviating the need for denaturing and remultiplication.

The transformed or transfected cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the selected host cells. A variety of suitable means are known in the art, including defined means and complex media and generally include a source of carbon, a source of nitrogen, essential amino acids, vitamins and minerals. The medium may also contain components such as growth factors or serum, as required. The growth medium will generally be selected for cells that contain the added DNA exogenously, for example, the selection or deficiency of drug in an essential nutrient which is complemented by the selectable marker carried out in the expression vector or co- transferred inside the host cell.

Zkun5 polypeptides, particularly short polypeptides, can also be prepared by chemical synthesis in accordance with methods known in the art, including solid phase synthesis, partial solid phase methods, fragmentary condensation or classical synthesis of solutions. See, for example, Merrifield, J. Am. Chem. Soc. 85: 2149, 1963; Stewart et al., Solid Phase Peptide Synthesis (2nd Edition), Pierce Chemical Co., Rockford, IL, 1984; Bayer and Rapp, Chem. Pept. Prot. 3: 3, 1986; and Atherton et al., Solid Phase Peptide Synthesis: A practical Approach (Synthesis of Solid Phase Peptides: A Practical Approach), IRL Press, Oxford, 1989.

It is preferred to purify the proteins of the present invention at a purity = 80%, more preferably = 90% purity, even more preferably = 95% purity, and particularly a pharmaceutically pure state, ie, greater than 99.9 is preferred. % purity with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, a purified protein is substantially free of other proteins, particularly other proteins of animal origin.

The zkun5 proteins are purified by conventional methods of protein purification, typically by a combination of chromatographic techniques. For example, polypeptides comprising a polyhistidine affinity aggregate (typically about 6 histidine residues) are purified by chromatographic affinity on a nickel chelate resin. See, for example, Houchuli et al., BiofTechnol. 6: 1321-1325, 1988.

Using methods known in the art, zkun5 proteins can be produced glycosylated or not glycosylated; pegylated or non-pegylated; and may or may not include an initial amino acid residue of methionine.

The zkun5 proteins are contemplated for the preparation of medicaments for use in the treatment or prevention of conditions associated with excessive proteinase activity, in particular an excess of trypsin, plasmin, kallikrein, elastase, cathepsin G, proteinase-3, thrombin, factor Vlla, factor IXa, factor Xa, factor Xla, factor Xlla, or matrix of metalloproteinases. Such conditions include, but are not limited to, acute pancreatitis, pulmonary injury with induced cardiopulmonary bypass (CPB), allergy-induced protease release, deep vein thrombosis, myocardial infarction, shock (including septic shock), hyperfibrinolytic hemorrhage, emphysema, arthritis rheumatoid, respiratory distress syndrome in adults, chronic inflammatory disease, psoriasis, and other inflammatory conditions. Medicines containing Zkun5 are also contemplated for use in the preservation of platelet function, organ preservation, and wound healing.

The proteins of Zkun5 may be useful in the treatment of conditions arising from an imbalance in hemostasis, including acquired coagulopathies, primary fibrinolysis and fibrinolysis due to cirrhosis, and complications of thrombolytic therapy by high doses. Acquired coagulopathies can result from liver diseases, uremia, acute disseminated intravascular coagulation, post-cardiopulmonary bypass, massive transfusion, or Warfarin overdose (Humphries, Transfusion Medici ne 1: 1181-1201, 1994). A deficiency or dysfunction in any of the procoagulant mechanisms predisposes the patient to spontaneous hemorrhage or excessive blood loss associated with trauma or surgery. Acquired coagulopathies usually involve a combination of deficiencies, such as deficiencies of a plurality of coagulation factors, and / or platelet dysfunction. In addition, patients with liver diseases commonly experience increased fibrinolysis due to the inability to maintain normal levels of 012-antiplasmin and / or decreased hepatic removal of plasminogen activators (Shuman, Hemorrhagic Disorders, in Bennet and Plu, editors, Cecil Textbook of Medicine (Cecil Textbook of Medicine), 20th edition, WB Saunders Co., 1996). Primary fibrinolysis results from a massive release of plasminogen activator. Conditions associated with primary fibrinolysis include carcinoma of the prostate, acute promyelocytic leukemia, hemangiomas, and sustained release of plasminogen activator by endothelial cells due to injection of poisons. Conditions become critical when sufficient plasmin is activated to reduce the circulating level of a2-antiplasmin (Shuman, cited above). The data suggest that plasmin in endothelial cells may be related to the pathophysiology of bleeding or re-thrombosis observed in patients undergoing high-level doppler thrombolytic therapy for thrombosis. Plasmin may cause additional damage to the thrombogenic surface of the blood vessels after thrombolysis, which may result in rethrombosis (Okajima, J. Lab. Clin. Med. 126: 1377-13 £? 4, nineteen ninety five) .

Additional antithrombotic uses of zkun5 proteins include treatment or prevention of deep vein thrombosis, pulmonary embolism, and post-surgical thrombosis.

The zkun5 proteins can be used in methods for the inhibition of coagulation in mammals, such as the treatment of disseminated intravascular coagulation. The zkun5 proteins can then be used in place of known anticoagulants such as heparin, coumarin, and antithrombin III. Such methods will generally include administration of the protein in an amount sufficient to produce clinically significant inhibition of blood coagulation. Such amounts will vary with the nature of the condition to be treated, but can be predicted based on known tests and experimental animal models, and will generally be within the ranges described below.

The proteins of zkun 5 can find a therapeutic use in blocking the degradation of proteolytic tissue. The proteolysis of extracellular matrix, connective tissue, and other tissues and organs is an element of several diseases. It is believed that this tissue destruction begins when plasmin activates one or more metalloproteinase matrices (eg, collagenase and metallo-elastases). The inhibition of plasmin by zku: 5 proteins can then be beneficial in the treatment of these conditions.

It is believed that metalloproteinase (MMPs) matrices play a role in the metastasis of cancers, abdominal aortic aneurysm, multiple sclerosis, rheumatoid arthritis, osteoarthritis, trauma and hemorrhagic shock, and corneal ulcers. MMPs produced by tumor cells break down and reshape tissue matrices during the process of metastatic spread. There is evidence to suggest that MMP inhibitors can block this activity (Brown, Advan, Enzyme Regul. 35: 293-301, 1995). Abdominal aortic aneurysm is characterized by degradation of the extracellular matrix and loss of structural integrity of the aortic wall, the data suggest that plasmin may be important in the sequence of events leading to this destruction of the aortic matrix ( Jean-Claude et al., Surgery 116: 472-478, 1994). It is also believed that proteolytic enzymes contribute to the inflammatory damage of multiple sclerosis tissue (Gijbels, J. Clin Invest 94: 2177-2182, 1994). Rheumatoid arthritis is a systemic, chronic inflammatory disease that predominantly affects joints and other connective tissues, where the proliferation of inflammatory tissue (panus) can cause deformities and joint dysfunctions (see, Arnett, in the Cecil Textbook of Medicine). Text Cecil de Medicina), cited above). Osteoarthritis is a chronic disease that causes the deterioration of the cartilage of the joint and other joint tissues and the formation of new bones (bone spurs) in the margins of the joints. There is evidence that MMPs participate in the degradation of collagen in the osteoarthritic articular cartilage matrix. The inhibition of the MMPs results in the inhibition of the collagen removal of the cartilage matrix (Spirito, Inflam.Res.44 (sup.2): S131-S132), 1995; O'Byrne, Inflam. Res. 44_ (sup.2): S117-S118, 1995; Karran, Ann. Rheuma tic Disease 54: 662-669, 1995). The zkun5 proteins can also be useful in the treatment of trauma and hemorrhagic shock. The data suggest the administration of an MP inhibitor after hemorrhage improves the cardiovascular response, hepatocellular function, and microvascular blood flow in various organs (Wang, Shock 6: 377-382, 1996). Corneal ulcers that result in blindness manifest as a rupture of the collagenous stromal tissue. Damage due to thermal or chemical injuries to the corneal surface often results in a situation of chronic wound healing. There is direct evidence for the role of MMPs in the defects of the base of the membrane associated with failure in the re-epithelialization of the cornea or skin (Fini, Am. J. Pa thol. 149: 1287-1302, 1996 ).

The zkun5 proteins of the present invention can be combined with other therapeutic agents to increase the activity (e.g., antithrombotic or anticoagulant activity) of such agents. For example, a zkun5 protein can be used in combination with tissue plasminogen activator in thrombolytic therapy.

The doses of zkun5 proteins will vary according to the severity of the condition being treated and can vary from about 10 μg / kg to 10 mg / kg of body weight, preferably 100 μg / kg to 5 mg / kg, with greater preference of 100 μg / kg to 1 mg / mg. The proteins being formulated in a pharmaceutically acceptable carrier or vehicle. It is preferred to prepare them in a form suitable for injection or infusion, such as dilution with sterile water, an isotonic or glucose saline solution, or a similar vehicle. In an alternative, the protein can be packaged as a lyophilized powder, optionally in combination with a pre-measured diluent, and resuspended immediately before use. The pharmaceutical compositions may additionally include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent loss of protein on road surfaces, etc. The formulation methods are within the level of someone with experience in the art. See, Remington: The Science and Practice of Pharmacy (Remington: The Science and Practice of Pharmacy), Gennaro, editor, Mack Publishing Co., Easton, PA, 19th. edition, 1995.

Gene therapy provides an alternative therapeutic approach for the supply of zkun5 proteins. If a mammal has a mutated or absent zkun5 gene, a polynucleotide encoding a zkun5 protein can be introduced into the cells of the mammal. In one embodiment, a gene encoding a zkun5 protein is introduced in vivo into a viral vector. Such vectors include an attenuated or defective DNA virus, such as a herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like. Defective viruses, which completely or almost completely lack viral genes, are preferred. A defective virus is not an infectious virus after its introduction into a cell. The use of defective viral vectors allows administration to cells in a specific, localized area, without concern that the vector may infect other cells. Examples of particular vectors include, without limitation, a herpes simplex 1 defective virus (HSV1) (Kaplitt et al., Molec., Cell Neurosci.2: 320-30, 1991); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al., J. Clin. Invest. 90: 626-30, 1992; and a defective adeno-associated virus vector (Samulski et al., J. Virol., 61: 3096-101, 1987, Samulski et al., J. Virol. 63: 3822-8, 1989).

In another embodiment, a zkun5 polynucleotide can be introduced into a retroviral vector, as described, for example, by Anderson et al., U.S. Patent No. 5,399,346; Mann et al., Cell 33: 153, 1983; Terrin et al., US Patent No. 4,650,764; Temin • et al., U.S. Patent No. 4,980,289; Markowitz et al., J. Virol. 62: 1120, 1988; Temin et al., U.S. Patent No. 5,124,263; Dougherty et al., WIPO Publication No. WO 95/07358; and Kuo et al., Blood 82: 845, 1993. Alternatively, the vector can be introduced by lipofection in vivo using liposomes. Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Felgner et al., Proc.Na.I. Acad. Sci. USA £ 4: 7413-7, 1987; Mackey et al., Proc. Na ti, Acad. Sci. USA 85: 8027-31, 1988).

In a further embodiment, the target cells are removed from the body, and a vector is introduced into the cells as a plasmid of AD? helpless. The transformed cells are then re-implanted into the body. The vectors of AD? defenseless for gene therapy can be introduced into the desired host cells by means of methods known in the art, for example, transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation use of a gene trigger or use of a DNA vector transporter. See, for example, Wu et al., J. Biol. Chem. 267: 963-7, 1992; Wu et al., J. Biol. Chem. 263: 14621-4, 1988.

The zkun5 proteins can also be used to prepare antibodies that specifically bind to zkun5 proteins. As used herein, the term "antibodies" includes polyclonal antibodies, monoclonal antibodies, their antigen binding fragments such as F (abA2 and Fab, single chain antibodies, and the like, including engineered antibodies. humans can be humanized by grafting only non-human CDRs into human tissue and constant regions, or by incorporating the entire non-human variable domains (optionally by "hiding" them with a human-like surface by replacement of exposed residues, wherein the result it is a "disguised" antibody.) In some cases, humanized antibodies can retain non-human residues in the variable region tissue domains to enhance the appropriate binding characteristics.As humanized antibodies, biological half-life is increased, and reduced the potential for adverse immune reactions in the administration to humans. No experience in the art can generate humanized antibodies with specific and different constant domains (ie, different Ig subclasses) to facilitate or inhibit various immune functions associated with particular domains of particular antibody. Alternative techniques for the generation or selection of antibodies useful herein include in vitro exposure of lymphocytes with a zkun5 protein, and selection of libraries for displaying antibodies in Fag vectors or the like (eg, by the use of immobilized zkun5 polypeptide or marked). Antibodies are defined as being specifically binding if they bind to a zkun5 protein with an affinity of at least 10 times greater than the binding affinity of the control polypeptide (not zkun5). It is preferred that the antibodies exhibit a binding affinity (Ka) of 106 M-1 or greater, preferably of 107 M 1 or greater, more preferably 108 M-1 or greater, much more preferably 109 M-1 or greater. . The affinity of a monoclonal antibody can be readily determined by one of ordinary skill in the art (see, for example, Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949).

Methods for the preparation of polyclonal and monoclonal antibodies are well known in the art (see for example, Hurrell, JGR, editor, Monoclonal Hybridoma Antibodies: Techniques and Applications (Monoclonal Hybridoma Antibodies: Techniques and Applications), CRC Press, Inc. Boca Ratón, FL, 1982). For someone with ordinary skill in the art, it will be apparent that polycyclic antibodies can be generated from a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice and rats. The immunogenicity of a zkun5 protein can be increased by the use of an adjuvant such as alum (aluminum hydroxide) or complete or incomplete Freund's adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as fusions of a zkun5 protein or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. The polypeptide immunogen can be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like", that portion may advantageously bind or bind to a macromolecular carrier (such as keyhole limpet hemocyanin).

(KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.

The immunogenic zkun5 polypeptides can be as small as 5 residues. It is preferred to use polypeptides that are hydrophilic or that comprise a hydrophilic region. One of those preferred regions of the SEC. DO NOT. IDENTIFICATION: 2 includes residues 43-59.

A variety of assays known to those skilled in the art can be used to detect antibodies that specifically bind to a zkun5 protein. Exemplary assays are described in detail in Antibodies: A Laboratory Manual (Antibodies: A Laboratory Manual), Harlow and Lane (Editors), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include concurrent immunoelectrophoresis, radioimmunoassays, radio-in unoprecipitations, enzyme-linked immunosorbent assays (ELISA), spot spot assays, Western spotting assays, inhibition or competition tests, and sandwich tests.iVE Antibodies to zkun5 can be used for affinity purification of zkun5 proteins; in diagnostic tests to determine zkun5 protein circulation levels; for the detection or quantification of soluble zkun5 protein as a marker of a hidden pathology or condition; for immunolocalization in all animals or tissue sections, including immunodiagnostic applications; for immunohistochemistry; for The discrimination of expression libraries; and for other uses that will be evident to those skilled in the art. Pa to certain applications, including diagnostic uses in vit ro .AiW ^ _ ^ _ ^ __ ^! G ^^^ 3 ^ and in vivo, it is advantageous to use labeled antibodies. Suitable aggregates or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminescent labels, magnetic particles and the like; among the aggregates or indirect markers can highlight the use of biotin-avidin or other complement / anti-complement pairs as intermediaries.

The zkun5 proteins can be used in the laboratory or commercial preparation of proteins from cultured cells. The proteins can be used alone to inhibit specific proteolysis or can be combined with other proteinase inhibitors to provide a "cocktail" with a broad spectrum of activity. Of particular interest is the inhibition of cellular proteases, which can be released during cell disruption. The proteins can also be used in the laboratory as a tissue culture additto prevent cell separation.

The present invention also provides reagents for use in diagnostic applications. For example, the zkun5 gene, a sample comprising DNA or RNA from zkun5 or its subsequent ones can be used to determine if the zkun5 gene is present on chromosome 7 or if a mutation has occurred. Chromosomal aberrations detectable at the zkun5 gene site include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements. Such aberrations can be detected using polynucleotides of the present invention employing molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, short row repeat (STR) analysis using PCR techniques, and other assay techniques. genetic linkage known in the art (Sambrook et al., cited above, Ausubel et al., cited above, Marian, Chest 108: 255-65, 1995).

The zkun5 polynucleotides can also be used for chromosome mapping. The human zkun5 gene has been located in 7p22.2-p22.1. The location of the zkun5 gene facilitates the establishment of directly proportional physical distances between newly discovered genes of interest and previously plotted markers, including zkun5. Accurate knowledge of the position of the gene can be useful for numerous purposes, including: 1) determination of whether a newly identified sequence is part of a gene or gene segment previously identified and obtaining additional surrounding genetic sequences in various forms, such as YACs, BACs or cDNA clones; 2) provision of a possible candidate gene for a hereditary disease that shows a link to the same chromosomal region; and 3) cross-referencing model organisms, such as mice, that can help determine what function a particular gene might have. A useful technique in this respect is hybrid radiation tracing, a somatic cell genetic technique developed to construct contiguous high-resolution maps of mammalian chromosomes (Cox et al., Science 250: 245-50, 1990). The partial or complete knowledge of the sequence of a gene allows us to design appropriate PCR primers for use with chromosomal radiation hybrid tracing panels. Hybrid radiation trace panels, which are commercially available (for example, the Stanford G3 RH Panel and the GeneBridge 4 RH Panel, available from Research Genetics, Inc. Huntsville, AL), completely cover the human genome. These panels allow the rapid localization and chromosomal ordering of genes based on the PCR, sites of sequences with aggregates (STSs), and other non-polymorphic and polymorphic markers within a region of interest.

Sites with labeled sequences (STSs) can also be used independently for chromosomal localization. An STS is a DNA sequence that is unique to the human genome and can be used as a reference point for a particular chromosome or region of a chromosome. An STS is defined by a pair of oligonucleotide primers that are used in the polymerase chain reaction to specifically detect this site in the presence of all other genomic sequences. Since STSs are based only on the DNA sequence, they can be fully described in an electronic database (for example, Database of Sequence Tagged Sites (dbSTS), Gen Bank, National Center for Biological Information, National Institutes of Health, Bethesda, MD, http://www.ncbi.nlm.nih.gov) and can be searched with a gene sequence of interest for the map data contained in these short STS signal sequences genomics The present invention is further illustrated by means of the following non-limiting examples.

EXAMPLES Example 1 Nucleotide sequence 139 of an expressed sequence tag or aggregate (EST) was analyzed and found to encode a C-terminal portion of a Kunitz domain. A clone corresponding to this EST was obtained and sequenced. The clone contained the sequence shown in SEC NO. IDENTIFICATION: 1. This Kunitz domain sequence was designated as 'zkun5'.

The tissue distribution analysis of zkun5 was performed by Northern blotting (using Human Multiple Tissue Stains I, II, and III, and Master N-stains from Clontech Laboratories, Inc., Palo Alto, CA). , 3e produced a sample from a gel-purified EcoRI-Xhol fragment of the original clone of zkun5, and was radioactively labeled using commercially available labeling equipment (Rediprime ™ DNA Marking System, Amersham Corp., Arlington Heights, IL) according to the manufacturer's specifications. The sample was purified using a commercially available pressure column (NucTrap® column; Stratagene, La Jolla, CA, see Patent North American No. 5,336,412). A commercially available hybridization solution (ExpressHyb ™ Hybridization Solution; Clontech Laboratories, Inc., PaLo Alto, CA) was used for prehybridization and as a hybridization solution for the spots. Hybridization was carried out overnight at 65 ° C, and then the spots were washed in 2X SSC and 0.05% SDS at room temperature, followed by a wash in 0.1X SSC and 0.1% SDS at 55 ° C. . Two main transcripts were observed in the 7.5 kb and 5.0 kb sizes. The signals were present in various tissues, including the spinal cord, trachea, heart, colon, small intestine, stomach, placenta, skeletal muscle, kidney, pancreas, prostate, testis, thyroid, and adrenal gland. The RNA appeared to be subject to the tissue-specific splice event since the trachea, testicle, and placenta gave different band sizes with respect to the others.

Example 2 Based on the tissue distribution of Northern Spotting experiments (see Example 1), 5 'RACE was performed on cDNAs made from various tissues including pancreas, heart, stomach and testis. The cDNAs were prepared using commercially available equipment (Marathon ™ Amplification Kit for cDNA from Clontech Laboratories, Inc., Palo Alto, CA) and an oligo (dT) primer.

To amplify the zkun5 DNA, 5 μl each of 1/100 of diluted cDNAs, 20 pmol each of oligonucleotide primers ZC9739 (SEQ ID NO: 12) and ZC15, 999 (SEQ ID NO. 13), and 1 U of a 2: 1 mixture of ExTaq ™ DNA polymerase (TaKaRa Biomedicines) and Pfu DNA polymerase (Stratagene, La Jolla, CA) (ExTaqfPfu) in 25 μl reaction mixtures. The reaction was incubated at 94 ° C for 2 minutes; 25 cycles of 94 ° C and 15 seconds, 66 ° C for 20 seconds, and 72 ° C for 30 seconds; and an incubation of 1 minute at 72 ° C. 1 μl each of 1/100 of the first diluted PCR product was used as a standard for a nested PCR. 20 pmol each of oligonucleotide primers ZC9719 (SEQ ID NO: 14) and ZC15, 998 (SEQ ID NO: 15), and 1U of ExTaqfPfu were used in 25 μl mixtures. The mixtures were incubated at 94 ° C for 2 minutes; 2 cycles of 94 ° C and 15 seconds, 66 ° C for 20 seconds, 72 ° C for 30 seconds; 25 cycles of 94 ° C and 15 seconds, 64 ° C for 20 seconds, 72 ° C for 30 seconds; and an incubation of 1 minute at 72 ° C. The PCR products were gel purified and sequenced. The sequencing results indicated that the PCR products are expanded more than the original EST clone to include an intact Kunitz domain. The sequence of the clone generated by PCR is shown in SEC. DO NOT. IDENTIFICATION: 9.

To build an expression vector for the domain Kunitz zkun5 PCR was performed on cDNA prepared from the pancreas as described above. Based on the comparison of domain with other known Kunitz domains, the primers were designed in such a way that the PCR product could encode an intact Kunitz domain with Bam Hl restriction sites in the sense primer ZC17,238 (SEC. IDENTIFICATION: 16) and Xho I in the antisense initiator ZC17,240 (SEQ ID NO: 17) to facilitate subcloning in an expression vector. A silent mutation (nucleotide T to C) was introduced into the sense primer ZC17,238 (SEQ ID NO: 16) to remove an internal Bam Hl site in the Kunitz domain sequence. 5 μl of diluted 1/100 cDNA, 20 pmol each of oligonucleotide primers ZC17,238 (SEQ ID NO: 16) and ZC17,240 (SEQ ID NO: 17), and 1 U of ExTaqfPfu in 25 μl reaction mixtures. The mixtures were incubated at 95 ° C for 2 minutes; 3 cycles of 94 ° C for 30 seconds, 50 ° C for 30 seconds, 72 ° C for 30 seconds; 35 cycles of 94 ° C and 30 seconds, 68 ° C for 30 seconds; and an incubation of 7 minutes at 72 ° C. The PCR product was purified by gel and a restricted digestion was carried out with Bam Hl and Xho I overnight.

A mammalian expression vector was constructed with the dihydrofolate reductase gene selection marker under control conditions of the first SV40 promoter, the SV40 polyadenylation site, and a cloning site to insert the gene of interest under the control of the metallothionein promoter (MT -1) of mouse and polyadenylation site hGH. The expression vector was designated pZP-9 and has been deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD under access no. 98688. To facilitate protein purification, the pZP9 vector was modified by the addition of a tissue plasminogen activator (t-PA) secretory signal sequence (see U.S. Patent No. 5,641,655) and a GluGlu aggregate sequence ( SEQ ID NO: 18) between the MT-1 promoter and the hGH terminator. The t-PA secretory signal sequence replaces the native signal sequence for the DNA encoding polypeptides of interest that are inserted into this vector, and the expression results in an N-terminal protein aggregate. The vector with added N-terminus was designated pZP9NEE. The vector was digested with Bam Hl and Xho I, and the zkun5 fragment was inserted. The resulting construction was confirmed by sequencing.

Example 3 The human zkun5 gene was mapped to chromosome 7 using the commercially available GeneBridge 4 Hybrid Radiation Panel (Research genetics, Inc., Huntsville, AL). The GeneBridge 4 Hybrid Radiation Panel contains DNAs capable of PCR from each 93 hybrid radiation clones, plus two control DNAs (the HFL donor and the A23 receptor). A server of the worldwide network available to the public (http: //www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl) allows the layout relative to the Whitehead Institute / MIT Center so that the Genome Research radiation traces the map of the human genome (the hybrid radiation map "WICGR"), which was constructed with the Hybrid Radiation Panel GeneBridge 4.

To trace the zkun5 gene, 20 μl reaction mixtures were placed in a 96-well microtiter plate with PCR capacity (Stratagene, La Jolla, CA) and used in a thermal cycle (RoboCycler® Gradiente 96; Strategen ). Each of the 95 ECR reaction mixtures contained 2 μl of buffer solution (Klen Taq 10X PCR reaction buffer, Clontech Laboratories, In. Palo Alto, CA), 1.6 μl of dNTPs mixtures (2.5 mM each, PERKIN-ELMER , Foster City, CA), 1 μl of sense initiator (ZC16,523; SEC. DO NOT. IDENTIFICATION: 19), 1 μl of antisense initiator (ZC16,522; SEQ ID NO: 20), 2 μl of a density-increasing agent and a screening dye (RediLoad, Research Genetics, Inc., Huntsville, AI), 0.4 μl of a commercially available mixture of DNA / antibody polymerase (50X Advantage ™ KlenTaq Polymerase Blend, Clontech Laboratories, Inc.), 25 ng of DNA from a single hybrid or control clone and x μl of ddH20, for a total volume of 20 μl. The mixtures were covered with an equal amount of mineral oil and sealed. The conditions of the PCR cycler were as follows: an initial denaturation of 5 minutes at 95 ° C; 35 cycles of a denaturation of 1 minute at 95 ° C, one minute of annealing at 64 ° C, and an extension of 1.5 minutes at 72 ° C; followed by a final extension of 7 minutes at 72 ° C. the reaction products were separated by electrophoresis on a 2% agarose gel (Life Technologies, Gaithersburg, MD).

The results showed that the zkun5 gene draws 2.74 cR_3000 from the structure marker D7S481 on the hybrid chromosomal radiation 7 WICGR map. The near and distance markers were D7S481 and CHLC.GATA84AO8, respectively. The use of surrounding markers places the zkun5 gene in the 7p22.2-p22.1 region on the integrated map of the LDB 7 chromosome (The Genetic Location Database), University of Southhampton, WWW server: http : //cedar.genetics.soton.ac.uk/ public_html /).

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the essence and scope of the invention. Accordingly, the invention is not limited except by the appended claims.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, the content of the following is claimed as property.

LIST OF SEQUENCES < 110 > ZymoGenetlcs. Inc. < 120 > DOMAIN POLIPEPTIDE OF KUNITZ AND MATERIALS AND METHODS FOR ITS DEVELOPMENT < 130 > 98-22PC < 150 > US 09 / 086,253 < 151 > 1998-05-28 < 160 > 20 < 170 > FastSEQ for Windows Version 3.0 < 210 > 1 < 211 > 186 < 212 > DNA < 213 > Homo sapiens < 220 > '< 221 > CDS < 222 (D ... Ü86) 400 > 1 gtg gat ggg gca gag gac ect aga tgt ttg gaa gcc ttg aag ect gga 48 Val Asp Gly Ala Glu Asp Pro Arg Cys Leu Glu Ala Leu Lys Pro Gly 1 5 10 15 aac tgt ggt gaa tat gtg gtt cga tgg tat tat gac aaa cag gtc aac 96 Asn Cys Gly Glu Tyr Val Val Arg Trp Tyr Tyr Asp Lys Gln Val Asn 20 25 30 tct tgt gcc cga ttt tgg ttc agt ggc tgt aat ggc tea gga aat aga 144 Ser Cys Wing Arg Phe Trp Phe Ser Gly Cys Asn Gly Ser Gly Asn Arg 35 40 45 ttc aac agt gaa aag gaa tgt ca gaa acc tgc att ca gga 186 Phe Asn Ser Glu Lys Glu Cys Gln Glu Thr Cys lie Gln Gly 50 55 60 < 210 > 2 < 211 > 62 < 212 > PRT < 213 > Homo sapiens < 400 > 2 Val Asp Gly Ala Glu Asp Pro Arg Cys Leu Glu Ala Leu Lys Pro Gly 1 5 10 15 Asn Cys Gly Glu Tyr Val Val Arg Trp Tyr Tyr Asp Lys Gln Val Asn 20 25 30 Ser Cys Ala Arg Phe Trp Phe Ser Gly Cys Asn Gly Ser Gly Asn Arg 35 40 45 Phe Asn Ser Glu Lys Glu Cys Gln Glu Thr Cys He Gln Gly 50 55 60 < 210 > 3 < 211 > 186 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > engineering variant < 221 > CDS < 222 > (1) ... (186) < 221 > mise feature < 222 > (1) .T. (186) < 223 > n - A.T.C or G < 400 > 3 gtg gat ggg gca gag gac ect aga tgt ttg gaa gcc ttg aag ect gga 48 Val Asp Gly Ala Glu Asp Pro Arg Cys Leu Glu Ala Leu Lys Pro Gly 1 5 10 15 aac tgt ggt gen tat gtg gtt cga tgg tat tat gac aaa cag gtc aac 96 Asn Cys Gly Ala Tyr Val Val Arg Trp Tyr Tyr Asp Lys Gln Val Asn 20 25 30 tct tgt gcc cga ttt tgg ttc agt ggc tgt aat ggc tea gga aat aga 144 Ser Cys Wing Arg Phe Trp Phe Ser Gly Cys Asn Gly Ser Gly Asn Arg 35 40 45 ttc aac agt gaa aag gaa tgt caa gaa acc tgc att ca gga 186 Phe Asn Ser Glu Lys Glu Cys Gln Glu Thr Cys He Gln Gly 50 55 60 < 210 > 4 < 211 > 62 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 engineering variant < 400 > 4 Val Asp Gly Ala Glu Asp Pro Arg Cys Leu Glu Ala Leu Lys Pro Gly 1 5 10 15 Asn Cys Gly Ala Tyr Val Val Arg Trp Tyr Tyr Asp Lys Gln Val Asn 25 30 Ser Cys Wing Arg Phe Trp Phe Ser Gly Cys Asn Gly Ser Gly Asn Arg 40 45 Phe Asn Ser Glu Lys Glu Cys Gln Glu Thr Cys He Gln Gly 50 55 60 < 210 > 5 < 211 > 51 < 212 PRT < 213 > Artificial Sequence < 220 > < 223 > polypeptide motif < 221 > VARIANT < 222 > (2) . . . (2) < 223 Xaa is any residue except Asp. Cys. Gly. H1s. Met. Pro. Trp or Val < 221 > VARIANT < 222 > (3) . . . (3) < 223 > Xaa 1s Leu. Glu. Met. Gln. Phe. Ser. Thr. Ala or Pro < 221 > VARIANT < 222 > (4) . . . (4) < 223 > Xaa is any residue except Arg. Cys. Met. Phe. Trp. Tyr or Val < 221 > 'VARIANTE < 222 > (5) ~. . (5) < 223 Xaa is any residue except Asn. Cys. Gln. Gly. Phe.

Ser. Thr or Trp < 221 > VARIANT < 222 > (6) ... (6) < 223 > Xaa is Arg. Glu. Asn. To. Val. Asp Lys. Ser. Tyr or Met < 221 > VARIANT < 222 > (7) ... (7) < 223 > Xaa is any residue except Asn. Cys, Gly. H1s. Leu Met. Phe or Trp < 221 > VARIANT < 222 > (8) ... (8) < 223 > Xaa is Gly or Glu < 221 > VARIANT < 222 > (9) ... (9) < 223 > Xaa 1s Pro. Arg. Leu. Val. Ser. Asp. He. Asn or Thr < 221 > VARIANT < 222 > (11) ... (11) < 223 > Xaa is any residue except Ala. Cys. Glu. His. He. Pro. Trp and Val < 221 > VARIANT < 222 > (12) ... (12) < 223 > Xaa 1s Arg. Lys. To. Asp Gln. Phe. Gly. Glu. Thr and I Ser < 221 > VARIANT < 222 > (13) ... (13) < 223 > Xaa is any residue except Asp. Cys. Glu. Pro. Thr or Trp < 221 > VARIANT < 222 > (14) ... (14) < 223 > Xaa is ualquietiBsiduo except Arg. Asn. Cys, Gly, H1s. Ser, Trp or Tyr < 221 > VARIANT < 222 > (15) ... (15) < 223 > Xaa is any residue except Ala. Asp Cys. Gly, His. Met. Trp or Tyr < 221 > VARIANT < 222 > (16).? (16) < 223 > Xaa (ies Ser. Ala. Arg. Val. Gln. Lys, Leu. Gly or He. <221> VARIANT <222> (17) ... (17) <223> Xaa rfs Phe. Tyr. He. Trp or Leu <221> VARIANT <222 (18) .- (18) <223> Xaa (ies) tyr, His. Phe. Trp. Asn or Ala <221> VARIANT < 222 > (19) ... (19) < 223 > Xaa (Í Tyr or Phe < 221 > VARJAN.TE < 222 (20 ^ (20) < 223 > XaaAs iys, Asn. Ser or Asp < 221 > VARIANTE < 222 > (21) ... (21) 223 > Xaa is any residue except Asp, Cys. Glu. His or Tyr < 221 > VARIANT < 222 > (22) ... (22) < 223 > Xaa is any residue except Cys. Met. Pro or Trp < 22i > VARIANT < 222 (23) ... (23) < 223> Xaa ^ Ala. Lys. Ser. Leu. Thr. He. Gln. Glu. Tyr or Val As <221> VARIANT <222> (24) ... (24) <223 Xaa @Lys Gln. Asn. His. Gly. Arg or Met < 22l5 VARIANT < 222 > (25) ... (25) < 223 > Xaa is any residue except Asn. Asp Cys. His. He. Pro. Trp Tyr or Val < 221 > VARIANT < 222 > (27) ... (27) < 223 > Xaa is any residue except cys. Gly. Phe. Pro. Ser or Trp < 221 > VARIANT < 222 > (28) ... (28) < 223 > Xaa is any residue except Asp. Cys. His. He. Phe. Trp or Tyr < 221 > VARIANT < 222 > (29) ... (29) < 223 > Xaa is Phe or Tyr < 221 > VARIANT < 222 > (30) ... (30) < 223? Xaa is any residue except Arg. Cys. Gly or Met < 221 > VARIANT < 222 > (31) ... (31) < 223 > Xaa is Tyr. Trp. Phe or Asp < 221 > VARIANT < 222 > (32) ... (32) < 223 > Xaa is Ser. Gly or Thr < 221 > VARIANT < 222 > (33) ... (33) < 223 Xaa is Gly or He < 221 > VARIANT < 222 > (35) ... (35) < 223 > 'Xaaes Gly. Lys. Arg. Pro. Gln. Leu Glu. Asn or Met < 221 > VARIANT < 222 > (36) ... (36) < 223 > .Xaaes Q] yt S or A1a < 221 > VARIANT < 222 > (37) ... (37) < 223 > Xaaes Asn. Lys or Ser < 221 > VARIANT < 222 > (38) ... (38) < 223 > Xaa is any residue except Cys. His. He. Phe. Pro. Thr. Trp.

Tyr or Val < 22? > VARIANT < 222 > (39) ... (39) < 223 Xaaes; Asn or Tyr < 221 > VARIANT < 222 > (40) ... (40) < 223 > Xaaes Arg. Asn. Lys. Gln or Val < 22i > VARIANT < 222 > (41) ... (41) < 223 > Xaaes Phe. Tyr or Asp < 221 > VARIANT < 222 > (42) ... (42) < 223 > Xaa is any residue except Cys. Gln. Gly. Phe or Trp < 22? > VARIANT < 222 (43) ... (43) < 223 Xaaes Thr. Ser. Arg. Lys or Asp < 221 > VARIANT < 222 > (44) ... (44) < 223 > Xaaes He. Leu Trp. Arg. Lys. Thr. Glu. Ala, Gln or Val < 22? > VARIANT < 222 > (45) ... (45) < 223 > Xaaes Glu. Asp To. His. Met. Val. Gln. Lys. Arg or Pro < 221 > VARIANT < 222 > (46) ... (46) < 223 > Xaaes Glu Lys. Gln. Asp Ala or Tyr < 221 > VARIANT < 222 > (48) ... (48) < 223 = Xaa is any residue except Ala. Cys. Gly. Phe. Pro. Ser. Thr Trp or Tyr < 221 > VARIANT 222 > (49) ... (49) < 223 > Xaa is any residue except cys. He. Leu Met. Phe. Pro. Ser Tyr or Val < 221 > VARIANT < 222 > (50) ... (50) < 223 > Xaaes Thr. To. Val. He. Phe. Leu Met. Lys. Tyr or Arg < 400 > 5 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 35 40 45 Xaa Xaa Cys 50 < 210 > 6 < 211 > 186 < 2i2 > DNA < 213 > Artificial Sequence < 220 > < 223 > Degenerate DNA < 22l > miscjoaracteostica < 222 > (1) ... (186) < 223 > n - A.T.C or G < 400 > 6 gtngayggng cngargaycc nmgntgyytn gargcnytna arccnggnaa ytgyggngar 60 taygtngtnm gntggtayta ygayaarcar gtnaaywsnt gygcnmgntt ytggttywsn 120 ggntgyaayg gnwsnggnaa ymgnttyaay wsngaraarg artgyearga racntgyath 180 carggn 186 < 210 > 7 < 211 > 186 < 212 DNA < 213 Artificial Sequence < 220 > < 223 > Degenerate DNA < 221 > mise Feature < 222 > (D.7.U86) < 223 > n - A.T.C or G < 400 > 7 gtngayggng cngargaycc nmgntgyytn gargcnytna arccnggnaa ytgyggngcn 60 taygtngtnm gntggtayta ygayaarcar gtpaaywsnt gygcnmgntt ytggttywsn 120 ggntgyaayg gnwsnggnaa ymntnthaay wsngaraarg artgyearga racntgyath 180 carggn 186 < 210 > 8 < 211 > 55 < 212 > PRT < 213 Homo sapiens < 400 > 8 Thr Asp He Cys Lys Leu Pro Lys Asp Glu Gly Thr Cys Arg Asp Phe 1 5 10 15 He Leu Lys Trp Tyr Tyr Asp Pro Asn Thr Lys Ser Cys Wing Arg Phe 20 25 30 Trp Tyr Gly Gly Cys Gly Gly Asn Glu Asn Lys Phe Gly Ser Gln Lys 35 40 45 Glu Cys Glu Lys Val Cys Ala 50 55 < 210 > 9 < 211 > 2383 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (1) ..., (1674) < 221 > mise feature < 222 > (1). *. (2383) < 223 > n - A.T.C or G < 400 > 9 ggg ccc gaa gga cea aag ggt gaa ccg ggc att atg ggc ect ttt gga 48 Gly Pro Glu Gly Pro Lys Gly Glu Pro Gly He Met Gly Pro Phe Gly 1 5 10 15 atg ect gga here tea ect gga cea ect ggg cea aga gga gat aga 96 Met Pro Gly Thr Ser He Pro Gly Pro Pro Gly Pro Lys Gly Asp Arg 20 25 30 gga gga ect ggg ata ect gga ttt aag gga gaa ect gga ctt tct att 144 Gly Gly Pro Gly He Pro Gly Phe Lys Gly Glu Pro Gly Leu Ser He 35 40 45 cga gga cea aag ggt gtc ca ggc ect cgg gga cea gtg ggt gct cea 192 Arg Gly Pro Lys Gly Val Gln Gly Pro Arg Gly Pro Val Gly Ala Pro 50 55 end. gga etc aaa ggt gat ggc tat ect ggt gtg ect gga ect cgt gga tta 240 Gly Leu Lys Gly Asp Gly Tyr Pro Gly Val Pro Gly Pro Arg Gly Leu. 65 70 75 80 cea gga ccc ect ggg ceg atg ggt tta cgt gga gtg gga gac act gga 288 Pro Gly Pro Pro Gly Pro Met Gly Leu Arg Gly Val Gly Asp Thr Gly 85 90 95 gca aag gga gag ect ggg gtc aga ggc ect cea ggt ect tct ggg ect 336 Wing Lys Gly Glu Pro Gly Val Arg Gly Pro Pro Gly Pro Ser Gly Pro 100 105 110 cgg ggc gta gga acc ggg aa ggt gat ggt gat act ggg cag aaa ggc 384 Arg Gly Val Gly Thr GVi Gly Pro Lys Gly Asp Thr Gly Gln Lys Gly 115 120 125 ttg ect ggc ect ect ggc ccc ect ggc tat gga tea cag gga att aaa 432 Leu Pro Gly Pro Pro Gly Pro Pro Gly Tyr Gly Ser Gln Gly He Lys 130 135 140 ggg gaa caga gga cea ca ggc gtc cea ggc cea aag ggc here atg ggc 480 Gly Glu Gln Gly Pro Gln Gly Pro Gly Pro Lys Gly Thr Met Gly 145 150 155 160 cat ggc etc cea ggc cag aag gga gag falls gga gaa cgg ggc gat gtg - 528 His Gly Leu Pro Gly Gln Lys Gly Glu His Gly Glu Gly Arg Gly Asp Val 165 170 175 gga aag aaa ggt gat aaa gga gaa att gga gag ect gga tct cea gga 576 Gly Lys Lys Gly Asp Lys Gly Glu He Gly Glu Pr or Gly Ser Pro Gly 180 185 190 aaa cag ggt tta ca gga ccc aaa gga gac cta gga ctt here aaa gaa 624 Lys Gln Gly Leu Gln Gly Pro Lys Gly Asp Leu Gly Leu Thr Lys Glu 195 200 205 gaa att ate aaa ctt att here gaa ata tgt ggt tgt ggg ccc aaa tgc 672 Glu He He Lys Leu He Thr Glu He Cys Gly Cys Gly Pro Lys Cys 210 215 220 aaa gag act cea cta gag ctg gtg ttt gtg ate gac age tea gaa age 720 Lys Glu Thr Pro Leu Glu Leu Val Phe Val He Asp Ser Ser Glu Ser 225 230 235 240 gtg ggg cea gag aac ttt cag ate att aaa aat ttt gtg aag act atg 76 Val Gly Pro Glu Asn Phe Gln He He Lys Asn Phe Val Lys Thr Met 245 250 255 gct gac cgg gtt gct ctg gac ctt gcc acg gcc cgc -ata ggc ata ate 81 <; Wing Asp Arg Val Wing Leu Asp Leu Wing Thr Wing Arg He Gly He He 260 265 270? UY aac tat age cat aag gtg gag aag gtg gct aat ttg aag cag ttc tcc 86-Asn Tyr Ser His Lys Val Glu Lys Val Wing Asn Leu Lys Gln Phe Ser 275 280 285 ?? t > age aag gat gac ttc aag ttg gct gta gac aac atg caa tat ctg ggg 91. Ser Lys Asp Asp Phe Lys Leu Wing Val Asp Asn Met Gln Tyr Leu Gly 290 295 300 gaa ggc here tac here gcc act gct ctg ca gca gcc aac gac atg ttt 96C Glu Gly Thr Tyr Thr Ala Thr Ala Leu Gln Ala Ala Asn Asp Met Phe 305 310 315 320 gaa gat gca agg cea ggt gta aaa aaa gtg gcc ttg gtc ate act gat 1008 Glu Asp Ala Arg Pro Gly Val Lys Lys Val Ala Leu Val He Thr Asp 325 330 335 gga cag here gat tct cgt gat aaa gag aaa ctg here gag gtg gtg aag 1056 Gly Gln Thr Asp Ser Arg Asp Lys Glu Lys Leu Thr Glu Val Val Lys 340 345 350 aat gcc agt gac acc aat gtg gag ata ttt gtg ata ggg gtg gtg aag 1104 Asn Ala Be Asp Thr Asn Val Glu He Phe Val He Gly Val Val Lys 355 360 365 aaa aat gat ccc aac ttt gaa ata ttc falls aaa gaa atg aat cta att 1152 Lys As Asp Pro Asn Phe Glu He Phe His Lys Glu Met Asn Leu He 370 375 380 gct act gac cea gag cat gtt tac cag ttt gat gat ttc ttt acc ctg 1200 Wing Thr Asp Pro Glu H1s Val Tyr Gln Phe Asp Asp Phe P he Thr Leu 385 390 395 400 caá gac acc ctg aag caa aaa ttg ttt caa aaa att tgt gag gat ttt 1248 Gln Asp Thr Leu Lys Gln Lys Leu Phe Gln Lys He Cys Glu Asp Phe 405 410 415 j ^ e &rj¿r ^^ í ^ ^ gattc tat etc gtt ca tt ttt ggt tea teg tea ect cact ect gga 1296 Asp Ser Tyr Leu Val Gln He Phe Gly Ser Ser Pro Pro Gln Pro Gly 420 425 430 ttt ggg atg tea ggg gaa gaa etc agt gaa tct act cea gag ect caa 1344 Phe Gly Met Ser Gly Glu Glu Glu Leu Ser Glu Ser Thr Pro Glu Pro Glu Pro 435 440 445 aaa gaa att tct gag tea ttg agt gtc acc aga gac cag gat gaa gat 1392 Lys Glu He Ser Glu Ser Leu Ser Val Thr Arg Asp Gln Asp Glu Asp 450 455 460 gat aag gct cea gag cea acg tgg gct gat gat cctct ect gcc act acc 1440 Asp Lys Wing Pro Pro Glu Pro Thr Trp Wing Asp Asp Leu Pro Wing Thr Thr 465 470 475 480 tea tct gag gcc acc action acc ccc agg cea ctg etc age acc ect gtg 1488 Be Ser Glu Wing Thr Thr Thr Pro Arg Pro Leu Leu Ser Thr Pro Val 485 490 495 gat ggg gca gag gat ect aga tgt ttg gaa gcc ttg aag ect gga aac 1536 Asp Gly Ala Glu Asp Pro Arg Cys Leu Glu Ala Leu Lys Pro Gly Asn 500 505 510 tgt ggt gaa tat gtg gtt cga tgg tat tat. gac aaa cag gtc aac tct 1584 Cys Gly Glu Tyr Val Val Arg Trp Tyr Tyr Asp Lys Gln Val Asn Ser 515 520 525 tgt gcc cga ttt tgg ttc agt ggc tgt aat ggc tea gga aat aga ttc 1632 Cys Ala Arg Phe Trp Phe Ser Gly Cys Asn Gly Ser Gly Asn Arg Phe 530 535 540 aac agt gaa aag gaa tgt caa gaa acc tgc att ca gga tga gcaagta 1681 Asn Ser Glu Lys Glu Cys Gln Glu Thr Cys He Gln Gly * 545 550 555 aattggcctg tetetatcaa aagcatagaa ctccctaatt tecacatatt cacccaatac 1741 ctatatttga aaatacagea gtgtatactg agtatttaca acttatacat gtaattgaat 1801 tctcactaca gccctaggat gtacatatta ttaaccactt atataggtaa gaaagctgag 1861 gctctgagaa gtttagtaac ttgtcaactg tcacccaact aaaaagtttc agagctgagg 1921 atttagactt agagctgtgt aacttcaata atetaettea cacagactct caacctgcaa 1981 tgtgattctg attcctttaa ttcctgttgt atgtactatg teagetcaaa cccctacccc 2041 tgtccctgcc catacctcca cccactcacc tccctaacct ccttatgtcc ctcacagtag 2101 caagatgtag gtgataggaa ggacttcggt gtgagaatta gaaatgatgt aaatgtttac 2161 gcaggagtgc tgggatagga gtcgggatgg tgagggtagt tagatttttg cctcacttgc 2221 cctgaaagtg gtaataggga gaaaccaatn tgaattacaa ttacttaaat gtatcacaga 2281 ctgtcacttt gtattcctcc aacatgtttg gtaacaagtg cttaatgtat gttaaaataa 2341 agaaggtttt tatacccttc cattaaaaaa aaaaaaaaaa aa 2383 < 210 > 10 < 211 > 557 < 212 PRT < 213 > Homo sapiens < 400 > 10 Gly Pro Glu Pro Gly Pro Lys Gly Pro Gly Pro Met Gly Pro Pro Ghe 1 5 10 15 Pro Met Pro Gly Pro Pro Pro Gly Pro Pro Gly Pro Gly Asp Ar Pro 20 25 30 Gly Gly Pro Pro Gly Pro Gly Pro Gly Glu Pro Gly Leu Be He 35 40 45 Arg Gly Pro Lys Gly Val Gln Gly Pro Arg Gly Pro Val Gly Wing Pro 50 55 60 Gly Leu Lys Gly Asp Gly Tyr Pro Gly Val Pro Gly Pro Arg Gly Leu 65 70 75 80 Pro Gly Pro Pro Gly Pro Met Gly Leu Arg Gly Val Gly Asp Thr Gly 85 90 95 Wing Lys Gly Glu Pro Gly Val Arg Gly Pro Pro Gly Pro Ser Gly Pro 100 105 110 Arg Gly Val Gly Thr Gln Gly Pro Lys Gly Asp Thr Gly Gln Lys Gly 115 120 125 Leu Pro Gly Pro Pro Gly Pro Pro Gly Tyr Gly Ser Gln Gly He Lys 130 135 140 Gly Glu Gln Gly Pro Gln Gly Phe Pro Gly Pro Lys Gly Thr Met Gly 145 150 155 160 His Gly Leu Pro Gly Gln Lys Gly Glu H1s Gly Glu Arg Gly Asp Val 165 170 175 Gly Lys Lys Gly Asp Lys Gly Glu He Gly Glu Pro Gly Ser Pro Gly 180 185 190 Lys Gln Gly Leu Gln Gly Pro Lys Gly Asp Leu Gly Leu Thr Lys Glu 195 200 205 Glu He He Lys Leu He Thr Glu He Cys Gly Cys Gly Pro Lys Cys 210 215 220 Lys Glu Thr Pro Leu Glu Leu Val Phe Val He Asp Ser Ser Glu Ser 225 230 235 240 Val Gly Pro Glu Asn Phe Gln He He Lys Asn Phe Val Lys Thr Met 245 250 255 Wing Asp Arg Val Wing Leu Asp Leu Wing Thr Wing Arg He Gly He He 260 265 270 Asn Tyr Ser His Lys Val Glu Lys Val Wing Asn Leu Lys Gln Phe Ser 275 280 285 Ser Lys Asp Asp Phe Lys Leu Wing Val Asp Asn Met Gln Tyr Leu Gly 290 295 300 Glu Gly Thr Tyr Thr Ala Thr Ala Leu Gln Ala Ala Asn Asp Met Phe 305 310 315 320 Glu Asp Ala Arg Pro Gly Val Lys Lys Val Ala Leu Val He Thr Asp 325 330 335 Gly Gln Thr Asp Ser Arg Asp Lys Glu Lys Leu Thr Glu Val Val Lys 340 345 350 Asn Wing Being Asp Thr Asn Val Glu He Phe Val He Gly Val Val Lys 355 360 365 Lys Asn Asp Pro Asn Phe Glu He Phe H1s Lys Glu Met Asn Leu He 370 375 380 Thr Wing Asp Pro Glu His Val Tyr Gln Phe Asp Asp Phe Phe Thr Leu 385 390 395 400 Gln Asp Thr Leu Lys Gln Lys Leu Phe Gln Lys He Cys Glu Asp Phe 405 410 415 Asp Ser Tyr Leu Val Gln He Phe Gly Ser Ser Pro Pro Gln Pro Gly 420 425 430 Phe Gly Met Ser Gly Glu Glu Leu Ser Glu Ser Thr Pro Glu Pro Gln 435 440 445 Lys Glu He Ser Glu Ser Leu Ser Val Thr Arg Asp Gln Asp Glu Asp 450 455 460 Asp Lys Wing Pro Glu Pro Thr Trp Wing Asp Asp Leu Pro Wing Thr Thr 465 470 475 480 Be Ser Glu Wing Thr Thr Thr Pro Arg Pro Leu Leu Ser Thr Pro Val 485 490 495 Asp Gly Ala Glu Asp Pro Arg Cys Leu Glu Ala Leu Lys Pro Gly Asn 500 505 510 Cys Gly Glu Tyr Val Val Arg Trp Tyr Tyr Asp Lys Gln Val Asn Ser 515 520 525 Cys Wing Arg Phe Trp Phe Ser Gly Cys Asn Gly Ser Gly Asn Arg Phe 530 535 540 Asn Ser Glu Lys Glu Cys Gln Glu Thr Cys He Gln Gly 545 550 555 < 210 > 11 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > thrombin division site 400 > 11 Leu Val Pro Arg 1 < 210 > 12 < 211 > 27 < 212 > DNA < 213 Artificial Sequence < 220 > 223 = - Oligonucleotide Initiator ZC9739 400 > 12 ccatcctaat acgactcact atagggc / < 210 > 13 < 211 > 22 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide Initiator ZC15.999 < 400 > 13 gtggcaaggt ccagagcaac ce " < 210 > 14 < 211 > ? 3 < 2i2 > DNA < 213 > Artificial Sequence < 220 > < 223 > Initiator Oligonucleotide ZC9719 < 400 > 14 acteactata gggctcgagc ggc Z3 < 210 > 15 < 211 > 25 < 212 > DNA 213 'Artificial Sequence < 220 > < 223 > Oligonucleotide initiator 'ZC15.998 < 400 > 15 aacccggtca gccatagtct tcaca 25 < 210 > 16 < 211 > 32 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide Initiator ZC17.238 < 400 > 16 cgggatccgt ggatggggca gaggacccta ga 32 < 210 > 17 < 2U > 32 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide Initiator ZC17.240 < 400 > 17 ccctcgagtc atccttgaat gcaggtttct tg 32 < 210 > 18 < 211 > 6 < 212 > PRT < 213 Artificial Sequence < 220 > < 223 > peptide aggregate < 400 > 18 Glu Tyr Met Pro Met Glu 1 5 < 210 > 19 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Initiator Oligonucleotide ZC16.523 00 19 agccttgaag cctggaaact 20 < 210 > 20 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide Initiator ZC16.522 < 400 > 20 agggagttet atgctttttg to 21

Claims (1)

1. CLAIMS An isolated protein, characterized in that it comprises a sequence of amino acid residues as shown in SEQ. DO NOT. IDENTIFICATION: 5, wherein said sequence is at least 90% identical to residues 9 to 59 of the SEC. DO NOT. OF IDENTIFICATION: 2 and wherein said protein possesses a proteinase inhibitory activity. The isolated protein in accordance with the claim 1, characterized in that said sequence is selected from the group consisting of: (a) residues 9 to 59 of the SEC. DO NOT. FROM IDENTIFICATION: 2; and (b) Sections 9 to 59 of the SEC. DO NOT. OF IDENTIFICATION: 4 The isolated protein according to claim 1 or claim 2, characterized in that said protein is of a length of 51 to 81 amino acid residues. The isolated protein according to claim 1 or claim 2, characterized in that said protein is of a length of 51 to 67 residues. 1, characterized in that it consists of contiguous amino acid residues 51-557 of the SEC. DO NOT. IDENTIFICATION: 10. The isolated protein according to claim 1 or claim 2, characterized in that it additionally comprises an affinity tag or aggregate. The isolated protein according to claim 6, characterized in that said affinity aggregate is maltose binding protein, polyhistidine, or Glu-Tyr-Met-Pro-Met-Glu (SEQ ID NO: 18). An expression vector comprising the following operable link elements: (a) a transcription promoter; (b) a DNA segment encoding a protein according to any of claims 1-7; and (c) a transcription terminator. The expression vector according to claim 8, characterized in that it additionally comprises a secretory signal sequence linked in operable form to the DNA segment. operable form to the DNA segment. A cultured cell containing an expression vector according to claim 8 or 9, characterized in that said cell expresses the DNA segment. A method for making a protein having a proteinase inhibitory activity, characterized in that it comprises: culturing the cell according to claim 10 under conditions by which said DNA segment is expressed; and recovery of the protein encoded by the DNA segment. An antibody that specifically binds to a protein as shown in SEQ. DO NOT. IDENTIFICATION: 2 or the SEC. DO NOT. IDENTIFICATION: 4. gg ^^ ggggs gf B2S2S £ ^^ SSL