Highly purified mocarhagin cobra venom protease polynucleotides endcoding same and related proteases and therapeutic uses thereof

Abstract

Highly purified mocarhagin, a cobra venom protease, is disclosed. Pharmaceutical compositions and therapeutic uses of the highly purified protease are also provided. Polynucleotides encoding such protease and related proteases are also disclosed.

Description

BACKGROUND OF THE INVENTION

Cellular interactions are key to many events in vascular biology. Cell surface adhesion molecules mediate many of the interactions between leukocytes, platelets and the vessel wall. In response to inflammatory stimuli, leukocytes and platelets in the adjacent vasculature initially roll on the blood vessel wall, then stick, and finally transmigrate to the site of insult. The initial rolling event involves a class of adhesion proteins termed selectins (P-, E-, and L-selectin) which mediate the interaction between leukocytes, platelets and endothelial cells by their recognition of specific carbohydrate counter-structures, including sialyl-Lewis x. The primary sequence/motif structure of each of the selectins is similar. Each contains a N-terminal, 118-amino acid calcium-dependent lectin domain, an EGF motif, a variable number of tandem repetitive motifs related to motifs found in complement regulatory domains, a transmembrane domain and a short cytoplasmic tail.

P-selectin is a 140-kDa integral granule membrane glycoprotein localized to the α-granules of platelets and the Weibel-Palade bodies of endothelial cells and is rapidly expressed on both cell types on cell activation. This suggests that endothelial P-selectin is a critical molecule mediating initial adhesion events in acute inflammation, a view recently supported by a number of in vivo inflammatory models including neutrophil-dependent acute lung injury (Mulligan et al. (1992) J. Clin. Invest. 90, 1600), endotoxin-induced neutropenia (Coughlan et al. (1994) J. Exp. Med. 179, 329), reperfusion injury (Asako et al. (1994) J. Clin. Invest. 93, 1508) and histamine-induced leukocyte rolling in post capillary venules (Weyrich et al. (1993) J. Clin. Invest. 91, 2620). P-selectin binds to 10,000-20,000 copies of a single class of binding sites on neutrophils and HL60 cells.

Sako et al. ((1993) Cell 75, 1179) have cloned a ligand for P-selectin, termed P-selectin glycoprotein ligand-1 (PSGL-1) found on the surface of leukocytes (see also copending application Ser. No. 08/316,305). PSGL-1 is a 220 kDa, disulfide-linked homodimeric sialomucin which, when expressed by recombinant methodology with the appropriate fucosyltransferase, binds P-selectin, E-selectin and L-selectin in a similar calcium-dependent manner to the PSGL-1 on neutrophils. PSGL-1 has a signal peptide sequence of 17 amino acids followed by a 24-amino acid PACE cleaved propeptide sequence. The mature N-terminus of PSGL-1 contains an unusual stretch of twenty amino acids which is rich in negatively-charged aspartate and glutamate residues and which contains three tyrosine residues which meet the consensus sequence for 0-sulfation by a golgi sulfotransferase. At least one of these tyrosine residues is sulfated as evaluated by site-directed mutagenesis (Sako et al.).

In addition to binding P-selectin, PSGL-1 also binds L- and E-selectin. In contrast to P-selectin, however, the requirements for E-selectin recognition are much less rigid. (Spertinit et al., J. Cell. Biol. 135:523 (1996)). E-selectin binds a wide variety of sialomucin structures if they co-express the sialyl-Lewis x structure. L-selectin binds to a number of different counter-receptors, GLYCAM-1, MadCAM-1 and CD34, which like PSGL-1, are also sialomucins. A major question currently unresolved is what determines selectin specificity in the recognition of specific counter-receptor structures. P-, E- and L-selectin are 60-70% homologous in their N-terminal, 118-amino acid lectin motifs and each similarly recognizes the sialyl-Lewis x and sialyl-Lewis a carbohydrate structures. Further, binding of P-selectin to its receptor on neutrophils is four to five orders of magnitude more avid than the binding of sialyl-Lewis x. While differences in specificity and avidity may in part be accounted for by either the presentation of multiple sialyl-Lewis carbohydrate structures on the receptor mucin core or by subtle differences in carbohydrate structure, it is probable that the protein component of the sialomucin also determines selectin interaction.

Although the inflammatory response mediated by the P-selectin/PSGL-1 interaction is a part of the body's normal defense system, inappropriate inflammatory responses can also result in the development of various inflammatory disease states. It would, therefore, be desirable to provide agents for interfering with or blocking the selectin/PSGL-1 interaction in order to treat inflammatory disease.

GP1bα is a component of the glycoprotein (GP) Ib-IX complex found on the surface of platelets and serving as a receptor for von Willibrand factor (vWF). The interaction of the GP IB-IX complex with vWF mediates attachment of platelets to the blood vessel wall at the site of injury. It has also can cause aggregation of platelets in high shear conditions and enable platelet activation at low concentrations of thrombin.

Mocarhagin, a protease found in the venom of cobras (including the Mozambiquan spitting cobra, Naja mossambica mossambica , a.k.a. Naja mocambique mocambique ), has been found to cleave PSGL-1, resulting in disruption of P- and L-selectin mediated cell adhesion. Preparations of mocarhagin have been reported and demonstrated to serve this purpose. See, U.S. Pat. No. 5,659,018; DeLuca et al., J. Biol. Chem. 270: 26734 (1995); Ward et al., Biochem. 35: 4929 (1996). (Spertini et al.)

In addition, it also has been reported that Mocarhagin is capable of cleaving GP1bα at a position proximal to sulfated tyrosine residues within the critical vWF binding domain and disrupting the binding activity of GP1bα: DeLuca et al., J. Biol. Chem. 270: 26734 (1995); Dong et al., Biochemistry, 33: 13946 (1994).

It is therefore anticipated that an agent that can disrupt this interaction may have therapeutic application in a variety of thrombotic disorders such as restenosis and DVT.

However, applicants have discovered that the preparations described in these documents is only partially purified. Since it is necessary for mocarhagin proteins to be provided in highly purified form for such proteins to be used for therapeutic purposes, it would be desirable to provide highly purified preparations of mocarhagin proteins.

It would also be desirable to identify and isolate polynucleotides encoding mocarhagin proteins in order to produce such proteins by recombinant methods.

SUMMARY OF THE INVENTION

The present invention provides compositions comprising a mocarhagin protein at least 95% free of other cobra proteins (preferably 95% free of all other proteins). Preferably, the mocarhagin is homogeneous (i.e., free of other proteins). In preferred embodiments, the mocarhagin protein is full-length mocarhagin (as described below). In other embodiments, the mocarhagin protein is a fragment of full-length mocarhagin having mocarhagin proteolytic activity. Preferably, the mocarhagin protein is characterized by at least one characteristic selected from the group consisting of:

(a) a molecular weight of approximately 55 kDa under reducing conditions;

(b) a molecular weight of approximately 55 kDa under nonreducing conditions;

(c) an N-terminal amino acid sequence comprising

TNTPEQDRYLQAKKYIEFYVVVDNVMYRKY (SEQ ID NO:1);

(d) mocarhagin proteolytic activity;

(e) the ability to inhibit platelet binding to vWF;

(f) requirement of calcium ion for activity;

(g) requirement of zinc ion for activity;

(h) an activity substantially inhibited by excess EDTA; and

(i) an activity substantially inhibited by high concentrations of DFP.

In some embodiments, the mocarhagin protein has the N-terminal sequences TNTPEQDRYLQAKKYIEFYVVVDNVMYRKYTGKLHVITXXVYEMNALN (SEQ ID NO:2).

In particularly preferred embodiments, the mocarhagin protein is capable of cleaving capable of cleaving a material selected from the group consisting of anionic polypeptides containing sulfated tyrosine residues, PSGL-1 and GP1bα. PSGL-1 and/or GP1bα. Compositions comprising a therapeutically effective amount of a mocarhagin protein and a pharmaceutically acceptable carrier are also provided.

Methods of treating an inflammatory disease and thrombotic disorders and of inhibiting selectin-mediated binding comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a mocarhagin protein to a mammalian subject are disclosed.

The invention also provides a method of isolating mocarhagin from venom, said method comprising:

(a) subjecting a composition comprising cobra venom to a heparin affinity chromatography column;

(b) subjecting the eluate from said heparin affinity column to a size exclusion column;

(c) subjecting the eluate from said size exclusion column to a Mono S column; and

(d) eluting said mocarhagin from said Mono S column.

Compositions comprising a protein isolated according to these methods (and optionally further comprising a pharmaceutically acceptable carrier) are also encompassed by the claimed invention. Such compositions can also be used in methods of treating an inflammatory disease and of inhibiting selectin-mediated binding which comprise administering a therapeutically effective amount of such compositions to a mammalian subject.

The present invention also provides a composition comprising a mocarhagin protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:

(a) the amino acid sequence of SEQ ID NO:6;

(b) the amino acid sequence of SEQ ID NO:6 from amino acid 24 to amino acid 621;

(c) the amino acid sequence of SEQ ID NO:6 from amino acid 192 to amino acid 621;

(d) fragments of the amino acid sequence of SEQ ID NO:6 encoding a protein having mocarhagin activity; and

(e) the amino acid sequence encoded by the cDNA insert of clone NMM-1 deposited under accession number ATCC 209588;

the protein being substantially free from other mammalian proteins.

Yet other embodiments provide for a composition comprising an isolated polynucleotide selected from the group consisting of:

(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5;

(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 78 to nucleotide 1940;

(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 147 to nucleotide 1940;

(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 651 to nucleotide 1940;

(e) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone NMM-1 deposited under accession number ATCC 209588;

(f) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6;

(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6 from amino acid 24 to amino acid 621;

(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6 from amino acid 192 to amino acid 621;

(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 encoding a protein having mocarhagin activity;

(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;

(k) a polynucleotide which encodes a species homologue of the protein of (f), (g) or (h) above; and

(l) a polynucleotide which hybridizes under stringent conditions to a polynucleotide of (a)-(h) above.

The present invention also provides a composition comprising a mocarhagin protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:

(a) the amino acid sequence of SEQ ID NO:8;

(b) the amino acid sequence of SEQ ID NO:8 from amino acid 24 to amino acid 439;

(c) the amino acid sequence of SEQ ID NO:8 from amino acid 192 to amino acid 439;

(d) fragments of the amino acid sequence of SEQ ID NO:8 encoding a protein having mocarhagin activity; and

(e) the amino acid sequence encoded by the cDNA insert of clone NMM-2 deposited under accession number ATCC 209589;

the protein being substantially free from other mammalian proteins.

Yet other embodiments provide for a composition comprising an isolated polynucleotide selected from the group consisting of:

(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7;

(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 85 to nucleotide 1401;

(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 154 to nucleotide 1401;

(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 658 to nucleotide 1401;

(e) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone NMM-2 deposited under accession number ATCC 209589;

(f) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8;

(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8 from amino acid 24 to amino acid 439;

(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8 from amino acid 192 to amino acid 439;

(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8 encoding a protein having mocarhagin activity;

(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;

(k) a polynucleotide which encodes a species homologue of the protein of (f), (g) or (h) above; and

(1) a polynucleotide which hybridizes under stringent conditions to a polynucleotide of (a)-(h) above.

The present invention also provides a composition comprising a mocarhagin protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:

(a) the amino acid sequence of SEQ ID NO:10;

(b) the amino acid sequence of SEQ ID NO:10 from amino acid 24 to amino acid 613;

(c) the amino acid sequence of SEQ ID NO:10 from amino acid 192 to amino acid 613;

(d) fragments of the amino acid sequence of SEQ ID NO:10 encoding a protein having mocarhagin activity; and

(e) the amino acid sequence encoded by the cDNA insert of clone NMM-9 deposited under accession number ATCC 209586;

the protein being substantially free from other mammalian proteins.

Yet other embodiments provide for a composition comprising an isolated polynucleotide selected from the group consisting of:

(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9;

(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 67 to nucleotide 1905;

(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 136 to nucleotide 1905;

(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 640 to nucleotide 1905;

(e) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone NMM-9 deposited under accession number ATCC 209586;

(f) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:10;

(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:10 from amino acid 24 to amino acid 613;

(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:10 from amino acid 192 to amino acid 613;

(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 encoding a protein having mocarhagin activity;

(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;

(k) a polynucleotide which encodes a species homologue of the protein of (f), (g) or (h) above; and

(l) a polynucleotide which hybridizes under stringent conditions to a polynucleotide of (a)-(h) above.

The present invention also provides a composition comprising a mocarhagin protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:

(a) the amino acid sequence of SEQ ID NO:12;

(b) the amino acid sequence of SEQ ID NO:12 from amino acid 24 to amino acid 521;

(c) the amino acid sequence of SEQ ID NO:12 from amino acid 192 to amino acid 521;

(d) fragments of the amino acid sequence of SEQ ID NO:12 encoding a protein having mocarhagin activity; and

(e) the amino acid sequence encoded by the cDNA insert of clone NMM-12 deposited under accession number ATCC 209585;

the protein being substantially free from other mammalian proteins.

Yet other embodiments provide for a composition comprising an isolated polynucleotide selected from the group consisting of:

(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:11;

(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:11 from nucleotide 78 to nucleotide 1640;

(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:11 from nucleotide 147 to nucleotide 1640;

(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:11 from nucleotide 651 to nucleotide 1640;

(e) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone NMM-12 deposited under accession number ATCC 209585;

(f) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:12;

(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:12 from amino acid 24 to amino acid 521;

(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:12 from amino acid 192 to amino acid 521;

(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:12 encoding a protein having mocarhagin activity;

(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;

(k) a polynucleotide which encodes a species homologue of the protein of (f), (g) or (h) above; and

(l) a polynucleotide which hybridizes under stringent conditions to a polynucleotide of (a)-(h) above.

The present invention also provides a composition comprising a mocarhagin protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:

(a) the amino acid sequence of SEQ ID NO:14;

(b) the amino acid sequence of SEQ ID NO:14 from amino acid 24 to amino acid 592;

(c) the amino acid sequence of SEQ ID NO:14 from amino acid 192 to amino acid 592;

(d) fragments of the amino acid sequence of SEQ ID NO:12 encoding a protein having mocarhagin activity; and

(e) the amino acid sequence encoded by the cDNA insert of clone NMM-13 deposited under accession number ATCC 209584;

the protein being substantially free from other mammalian proteins.

Yet other embodiments provide for a composition comprising an isolated polynucleotide selected from the group consisting of:

(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13;

(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13 from nucleotide 83 to nucleotide 1858;

(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13 from nucleotide 152 to nucleotide 1858;

(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13 from nucleotide 656 to nucleotide 1858;

(e) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone NMM-13 deposited under accession number ATCC 209584;

(f) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:14;

(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:14 from amino acid 24 to amino acid 592;

(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:14 from amino acid 192 to amino acid 592;

(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:14 encoding a protein having mocarhagin activity;

(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;

(k) a polynucleotide which encodes a species homologue of the protein of (f), (g) or (h) above; and

(l) a polynucleotide which hybridizes under stringent conditions to a polynucleotide of (a)-(h) above.

The present invention also provides a composition comprising a mocarhagin protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:

(a) the amino acid sequence of SEQ ID NO:16;

(b) the amino acid sequence of SEQ ID NO:16 from amino acid 62 to amino acid 462;

(c) fragments of the amino acid sequence of SEQ ID NO:16 encoding a protein having mocarhagin activity; and

(d) the amino acid sequence encoded by the cDNA insert of clone NMM-3 deposited under accession number ATCC 209587;

the protein being substantially free from other mammalian proteins.

Yet other embodiments provide for a composition comprising an isolated polynucleotide selected from the group consisting of:

(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15;

(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15 from nucleotide 3 to nucleotide 1388;

(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15 from nucleotide 186 to nucleotide 1388;

(d) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone NMM-3 deposited under accession number ATCC 209587;

(e) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:16;

(f) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:16 from amino acid 62 to amino acid 462;

(g) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:16 encoding a protein having mocarhagin activity;

(h) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;

(i) a polynucleotide which encodes a species homologue of the protein of (e) or (f) above; and

(j) a polynucleotide which hybridizes under stringent conditions to a polynucleotide of (a)-(g) above.

The present invention also provides a composition comprising a mocarhagin protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:

(a) the amino acid sequence of SEQ ID NO:18;

(b) the amino acid sequence of SEQ ID NO:18 from amino acid 197 to amino acid 621;

(c) fragments of the amino acid sequence of SEQ ID NO:18 encoding a protein having mocarhagin activity; and

(d) the amino acid sequence encoded by the cDNA insert of clone NMM-9ek deposited under accession number ATCC 209583;

the protein being substantially free from other mammalian proteins.

Yet other embodiments provide for a composition comprising an isolated polynucleotide selected from the group consisting of:

(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17;

(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17 from nucleotide 67 to nucleotide 1929;

(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17 from nucleotide 655 to nucleotide 1929;

(d) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone NMM-9ek deposited under accession number ATCC 209583;

(e) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:18;

(f) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:18 from amino acid 197 to amino acid 621;

(g) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:18 encoding a protein having mocarhagin activity;

(h) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;

(i) a polynucleotide which encodes a species homologue of the protein of (e) or (f) above; and

(j) a polynucleotide which hybridizes under stringent conditions to a polynucleotide of (a)-(g) above.

Compositions comprising an antibody which specifically reacts with the mocarhagin proteins or a fragments thereof having mocarhagin proteolytic activity are also provided.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 depicts an SDS-PAGE gel analysis of fractions containing mocarhagin eluted from the size exclusion column as described herein. Multiple protein species of similar molecular weight can be seen in these fractions.

FIG. 2 depicts an SDS-PAGE gel analysis of fractions containing mocarhagin eluted from the Mono-S column as described herein. This gel demonstrates the high degree of purity of the mocarhagin material purified as described in Example 1.

FIG. 3 is an SDS-PAGE gel analysis of fractions containing enterokinase-cleaved mocarhagin protein produced by expression of the NMM-9ek construct described below. The dot indicates the novel ˜50 k band produced by enterokinase cleavage.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

The present invention provides a highly specific metalloproteinase, mocarhagin, which has been purified from the venom of the Mozambiquan spitting cobra, Naja mossambica mossambica (a.k.a., Naja mocambique mocambique ). Mocarhagin cleaves a ten amino acid peptide from the mature N-terminus of PSGL-1 and abolishes the ability of PSGL-1 to bind P-selectin. These results are in accord with the negative charge/sulfated tyrosine cluster at the N-terminus of PSGL-1 being an important determinant of P-selectin recognition in addition to the recognition of carbohydrate structure.

Mocarhagin can be purified from cobra venom according to the method described in the examples below. Other methods of purifying mocarhagin from cobra venom will also be apparent to those skilled in the art. The progress of any purification scheme for mocarhagin can be monitored on the basis of the biochemical characteristics of mocarhagin described herein and the assays for PSGL-1 digestion and neutrophil/HL60 cell binding described below.

A cDNA encoding a mocarhagin protein (“clone NMM-1”) has also been cloned from a cobra venom gland library as described in Example 5 below. The nucleotide sequence of the NMM-1 cDNA is reported as SEQ ID NO:5. Clone NMM-1 was deposited with the American Type Culture Collection on Jan. 16, 1998 at accession number ATCC 209588. The protein sequence encoded by clone NMM-1 is reported as SEQ ID NO:6. Amino acids 1-23 of SEQ ID NO:6 are a predicted signal peptide. The mocarhagin propeptide begins with amino acid 24, with the mature protein beginning at amino acid 192.

Four additional full-length cDNAs encoding closely related proteases clones “NMM-2”, “NMM-9”, “NMM-12” and “NMM-13”) were also isolated from the cobra venom gland library as described in Example 5 below. Each of the proteins encoded by such cDNAs is also a “mocarhagin protein” as used herein.

The nucleotide sequence of the clone NMM-2 cDNA is reported as SEQ ID NO:7. Clone NMM-2 was deposited with the Americal Type Culture Collection on Jan. 16, 1998 at accession number ATCC 209589. The protein sequence encoded by clone NMM-2 is reported as SEQ ID NO:8. Amino acids 1-23 of SEQ ID NO:8 are a predicted signal peptide. The mocarhagin propeptide begins with amino acid 24, with the mature protein beginning at amino acid 192.

The nucleotide sequence of the clone NMM-9 cDNA is reported as SEQ ID NO:9. Clone NMM-9 was deposited with the Americal Type Culture Collection on Jan. 16, 1998 at accession number ATCC 209586. The protein sequence encoded by clone NMM-2 is reported as SEQ ID NO:10. Amino acids 1-23 of SEQ ID NO:10 are a predicted signal peptide. The mocarhagin propeptide begins with amino acid 24, with the mature protein beginning at amino acid 192.

The nucleotide sequence of the clone NMM-12 cDNA is reported as SEQ ID NO:11. Clone NMM-12 was deposited with the Americal Type Culture Collection on Jan. 16, 1998 at accession number ATCC 209585. The protein sequence encoded by clone NMM-12 is reported as SEQ ID NO:12. Amino acids 1-23 of SEQ ID NO:12 are a predicted signal peptide. The mocarhagin propeptide begins with amino acid 24, with the mature protein beginning at amino acid 192.

The nucleotide sequence of the clone NMM-13 cDNA is reported as SEQ ID NO:13. Clone NMM-13 was deposited with the Americal Type Culture Collection on Jan. 16, 1998 at accession number ATCC 209584. The protein sequence encoded by clone NMM-13 is reported as SEQ ID NO:14. Amino acids 1-23 of SEQ ID NO:14 are a predicted signal peptide. The mocarhagin propeptide begins with amino acid 24, with the mature protein beginning at amino acid 192.

Two additional partial cDNAs encoding other closely related proteases (clones “NMM-3” and “NMM-10”) were also isolated from the cobra venom gland library as described in Example 5 below. Each of the proteins ecnoded by such cDNAs is also a “mocarhagin protein” as used herein.

The nucleotide sequence of the clone NMM-3 cDNA is reported as SEQ ID NO:15. Clone NMM-3 was deposited with the Americal Type Culture Collection on Jan. 16, 1998 at accession number ATCC 209587. The protein sequence encoded by clone NMM-3 is reported as SEQ ID NO:16. Amino acids 1-61 of SEQ ID NO:16 are part of the propeptide sequence. The mature mocarhagin propeptide begins with amino acid 62.

Applicants have also discovered that removal of the mocarhagin propeptide increases the catalytic activity of the enzyme. Thus engineered recombinant forms of mocarhagin include forms having endopeptidase cleavage sites between the propeptide segment and mature peptide segment, including but not limited to, enterokinase cleavage sites or PACE cleavage sites. Alternatively, a propeptide or secretory signal peptide may be substituted for the native mocarhagin propeptide to enable the secretion of active recombinant mocarhagin from eucaryotic host cells.

The NMM-9 cDNA was used to make a modified construct which includes an enterokinase cleavage sight. Certain preferred embodiments of the present invention included such an enterokinase cleavage site in order to increase production of active (i.e., properly cleaved to remove the leader sequence) protein. The nucleotide sequence of the construct containing the celavage site, clone NMM-9ek, is reported as SEQ ID NO:17. Clone NMM-9ek was deposited with the Americal Type Culture Collection on Jan. 16, 1998 at accession number ATCC 209583. The protein sequence encoded by clone NMM-9ek is reported as SEQ ID NO:18. The enterokinase cleavage site is found at amino acid 192-196 of SEQ ID NO:18. Amino acids 1-196 of SEQ ID NO:18 are part of the propeptide sequence which is cleaved upon enterokinase treatment. The mature cleaved mocarhagin propeptide begins with amino acid 197.

For the purposes of the present invention, a protein is defined as having “mocarhagin proteolytic activity” when (1) it digests PSGL-1, such as in the PSGL-1 digestion assay described in Example 3 below, and/or (2) inhibits the binding of P-selectin to neutrophils or HL60 cells, such as in the binding inhibition assay described in Example 2 below, and/or (3) cleaves a peptide derived from PSGL-1 (pyroEATEYEYLDYDFLPE, SEQ ID NO:3), such as in the peptide cleavage assay described in Example 4 below. Preferably, in the PSGL-1 digestion assay complete cleavage of 35 [S]-sPSGL-1.T7 is achieved in 20 min. using 10 μg/mL mocarhagin protein; more preferably in 20 min. using less than 1 μg/ml mocarhagin protein. Preferably, in the neutrophil/HL 60 binding inhibition assay the mocarhagin protein exhibits an IC 50 of less than about 100 μg/mL, more preferably less than about 1 μg/mL.

Fragments of mocarhagin having mocarhagin proteolytic activity are also encompassed by the present invention. Fragments of mocarhagin having mocarhagin proteolytic activity can be identified by the PSGL-1 digestion assay and neutrophil/HL60 binding inhibition assay described below. Fragments of mocarhagin may be in linear form or they may be cyclized using known methods, for example, as described in H. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S. McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are incorporated herein by reference. For the purposes of the present invention, all references to “mocarhagin protein” herein include mocarhagin and fragments having mocarhagin proteolytic activity.

Isolated mocarhagin protein may be useful in treating conditions characterized by P- or E-selectin mediated intercellular adhesion or adhesion events mediated by GP1bα, including without limitation those involving platelet aggregation. Such conditions include, without limitation, myocardial infarction, vessel restenosis, thrombosis, bacterial or viral infection, metastatic conditions, inflammatory disorders such as arthritis, acute respiratory distress syndrome, asthma, emphysema, delayed type hypersensitivity reaction, systemic lupus erythematosus, thermal injury such as bums or frostbite, autoimmune thyroiditis, experimental allergic encephalomyelitis, multiple sclerosis, multiple organ injury syndrome secondary to trauma, diabetes, Reynaud's syndrome, neutrophilic dermatosis (Sweet's syndrome), inflammatory bowel disease, Grave's disease, glomerulonephritis, gingivitis, periodontitis, hemolytic uremic syndrome, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, granulocyte transfusion associated syndrome, cytokine-induced toxicity, and the like. Mocarhagin protein may also be useful in organ transplantation, both to prepare organs for transplantation and to quell organ transplant rejection. Mocarhagin protein may be used to treat hemodialysis and leukophoresis patients. Mocarhagin protein may be used itself as an inhibitor of P- or E-selectin-mediated intercellular adhesion or to design inhibitors of P- or E-selectin-mediated intercellular adhesion. The present invention encompasses both pharmaceutical compositions containing mocarhagin protein and therapeutic methods of treatment or use which employ mocarhagin protein.

Mocarhagin protein may be used as a pharmaceutical composition when combined with a pharmaceutically acceptable carrier. Such a composition may contain, in addition to mocarhagin protein and carrier, diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, G-CSF, Meg-CSF, stem cell factor, and erythropoietin. The pharmaceutical composition may contain thrombolytic or anti-thrombotic factors such as plasminogen activator and Factor VIII. The pharmaceutical composition may further contain other anti-inflammatory agents. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with mocarhagin protein, or to minimize side effects caused by the mocarhagin protein. Conversely, mocarhagin protein may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.

The pharmaceutical composition of the invention may be in the form of a liposome in which mocarhagin protein is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers which in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. No. 4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; and U.S. Pat. No. 4,737,323, all of which are incorporated herein by reference.

As used herein, the term “therapeutically effective amount” means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., healing of chronic conditions characterized by P-selectin- or E-selectin-mediated or GP1bα-mediated cellular adhesion or increase in rate of healing of such conditions. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

In practicing the method of treatment or use of the present invention, a therapeutically effective amount of mocarhagin protein is administered to a mammal having a P-selectin-mediated or GP1bα-mediated disease state. Mocarhagin protein may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors. When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, isolated mocarhagin protein may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering isolated mocarhagin protein in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.

Administration of mocarhagin protein used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, or cutaneous, subcutaneous, or intravenous injection. Intravenous administration to the patient is preferred.

When a therapeutically effective amount of mocarhagin protein is administered orally, mocarhagin protein will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% mocarhagin protein, and preferably from about 25 to 90% mocarhagin protein. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of mocarhagin protein and preferably from about 1 to 50% mocarhagin protein.

When a therapeutically effective amount of mocarhagin protein is administered by intravenous, cutaneous or subcutaneous injection, mocarhagin protein will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to mocarhagin protein an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additive known to those of skill in the art.

The amount of mocarhagin protein in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of mocarhagin protein with which to treat each individual patient. Initially, the attending physician will administer low doses of mocarhagin protein and observe the patient's response. Larger doses of mocarhagin protein may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.1 μg to about 100 mg of mocarhagin protein per kg body weight.

The duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each application of the mocarhagin protein will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention.

Mocarhagin protein of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specifically react with the mocarhagin protein and which may inhibit P-selectin-mediated or GP1bα-mediated cellular adhesion. Such antibodies may be obtained using the entire mocarhagin protein as an immunogen, or by using fragments of mocarhagin protein such as the soluble mature mocarhagin protein. Smaller fragments of the mocarhagin protein may also be used to immunize animals. The peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH). Additional peptide immunogens may be generated by replacing tyrosine residues with sulfated tyrosine residues. Methods for synthesizing such peptides are known in the art, for example, as in R. P. Merrifield, J.Amer.Chem.Soc. 85, 2149-2154 (1963); J. L. Krstenansky, et al., FEBS Lett. 211, 10 (1987).

Also included in the invention are isolated DNAs which hybridize to the DNA sequence set forth in SEQ ID NO:5 under stringent (e.g. 4×SSC at 65° C. or 50% formamide and 4×SSC at 42° C.), or relaxed (4×SSC at 50° C. or 30-40% formamide at 42° C.) conditions.

The isolated polynucleotides of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce the mocarhagin proteins recombinantly. Many suitable expression control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990). As defined herein “operably linked” means enzymatically or chemically ligated to form a covalent bond between the isolated polynucleotide of the invention and the expression control sequence, in such a way that the mocarhagin protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.

A number of types of cells may act as suitable host cells for expression of the mocarhagin protein. Suitable host cells are capable of attaching carbohydrate side chains characteristic of functional mocarhagin protein. Such capability may arise by virtue of the presence of a suitable glycosylating enzyme within the host cell, whether naturally occurring, induced by chemical mutagenesis, or through transfection of the host cell with a suitable expression plasmid containing a polynucleotide encoding the glycosylating enzyme. Host cells include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, or HaK cells.

The mocarhagin protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBac® kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), incorporated herein by reference.

Alternatively, it may be possible to produce the mocarhagin protein in lower eukaryotes such as yeast, fungi or in prokaryotes such as bacteria. Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe , Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium , or any bacterial strain capable of expressing heterologous proteins. Suitable fungi strains include Aspergillus sp.or any fungi strain capable of expressing heterologous proteins.

The mocarhagin protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a polynucleotide encoding the mocarhagin protein.

The mocarhagin protein of the invention may be prepared by culturing transformed host cells under culture conditions necessary to express a mocarhagin protein of the present invention. The resulting expressed protein may then be purified from culture medium or cell extracts as described in the examples below.

Alternatively, the mocarhagin protein of the invention is concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a purification matrix such as a gel filtration medium. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred (e.g., S-Sepharose® columns). The purification of the mocarhagin protein from culture supernatant may also include one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3GA Sepharose®; or by hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or by immunoaffinity chromatography.

Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the mocarhagin protein. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein. The mocarhagin protein thus purified is substantially free of other mammalian or other host cell proteins and is defined in accordance with the present invention as “isolated mocarhagin protein”.

EXAMPLES

The following examples are presented to illustrate, not to limit, the present invention.

Example 1

Purification of Mocarhagin

20 grams of crude protein from snake venom ( Naja mossambica mossambica , Sigma, product no. V1627) were dissolved in 500 mL deionized H 2 O and centrifuged at 10 K rpm for thirty minutes at 4 C. The supernatant was loaded onto a 200 mL Heparin-650 M affinity column (Toyopearl, Tosohaas) equilibrated with 50 mm Tris-HCl ph 7.6 (buffer A) and 0.2M NaCl. The column was first washed extensively (to baseline) and mocarhagin was eluted with a gradient of 0.2-1.0 M NaCI in buffer A. Fractions containing the protease as monitored by SDS-PAGE (band with molecular weight ˜55 kD) were pooled, concentrated using bentriprep-10 (Amicon) and applied to 21.5 mm ID×60 CM size exclusion column (G 3000SW, Tosohaas) in PBS at RT. Fractions eluted from the size exclusion column were analyzed by SDS-PAGE (FIG. 1 ), which showed the presence of multiple proteins of similar molecular weight.

Fractions containing mocarhagin were pooled and applied onto a Mono S 10/10 column (Pharmacia) equilibrated in 50 mm HEPES ph 8.0 (buffer B) at RT a O-IM NaCI in buffer B, gradient was used to elute the protein. The fractions were assayed by SDS-PAGE, pooled and frozen at 80° C. The recovery was 2-3 mg of mocarahagin per gram snake venom processed with a purity greater than 95%. FIG. 2 depicts a gel demonstrating the purity of the mocarhagin produced as herein described.

The N-terminal sequence was determined for the process described above as

TNTPEQDRYLQAKKYIEFYVVVDNVMYRKYTGKLHVITXXVYEMNALN (SEQ ID NO:2). The residues indicated in caps

(TNTPEQDRYLQAKKYIEFYVVVDNVMYRKY, SEQ ID NO:1) were determined to a higher degree of certainty.

Example 2

Neutrophil/HL60 Binding Inhibition Assay

Neutrophils were isolated from venous blood anticoagulated with heparin (20 units/mL, final concentration) according to the method of Bignold and Ferrante ((1987) J. Immunol. Meth. 96, 29). The neutrophils were >95% pure as evaluated by flow cytometry and >98% viable by trypan blue exclusion. HL60 cells were cultured in RPMI medium supplemented with 10% fetal calf serum. Immediately before use, cells were washed twice with phosphate-buffered saline (0.01 M sodium phosphate, 0.15 M sodium chloride, pH 7.4). Neutrophils and cultured cells were finally resuspended at 2×10 7 /mL in RPMI medium supplemented with 1% fetal calf serum. Binding of 125 I-labeled P-selectin (Skinner et al.) to neutrophils or HL60 cells w as evaluated by incubating 125 I-labeled P-selectin (0.5 μg/mL, final concentration) with cells (1×10 7 /mL, final concentration) at 22° C. in a final volume of 200 μl. After 30 min, duplicate 50 μl aliquots were withdrawn and loaded onto 200 μl of 17% (w/v) sucrose in RPMI medium containing 1% fetal calf serum. Neutrophils were pelleted at 8,750 ×g for 2 min. After careful aspiration of the supernatant, radiolabel associated with the cell pellets was measured in a-counter. Nonspecific binding of 125 I-labeled P-selectin was assessed using a 50-fold excess of unlabeled P-selectin (Skinner et al.).

To examine the effect of pretreatment of neutrophils or HL60 cells with mocarhagin on P-selectin binding, washed cells (2×10 7 /mL) in RPMI made 1% in fetal calf serum were incubated in the presence or absence of 10 mM EDTA followed by mocarhagin (0.025-100 μg/mL, final concentrations) for 30 min at 22C. P-selectin binding was then either directly assessed or was assessed after centrifugation of the cells, which were then washed twice and finally resuspended in RPMI with 1% fetal calf serum. In some experiments, DFP-treated mocarhagin was employed in place of mocarhagin. To evaluate the effect of supernatant from mocarhagin treated cells on P-selectin binding, HL60 cells at 10 8 /mL in 0.01 M Tris, 0.015 M sodium chloride, 0.001 M calcium chloride, pH 7.4, were incubated with mocarhagin (12 μg/mL) for 10 min at 22° C. The supernatant collected following centrifugation at 1000×g for 10 min was made 0.1% in BSA and loaded onto a heparin Sepharose CL-6B column (0.5×5 cm) to remove mocarhagin. The flow through was then tested for its effect on P-selectin binding to HL60 cells.

Example 3

PSGL-1 Digestion Assay

COS cells were cotransfected with three plasmids encoding soluble PSGL-1 (pED.sPSGL-1.T7; Sako et al.), alpha 1,3/1,4 fucosyltransferase (pEA.3/4FT) and soluble PACE (pEA-PACE SOL; Wasley et al. (1993) J. Biol. Chem. 268, 8458-8465). [ 35 S]Methionine-labeled COS conditioned medium containing sPSGL-1.T7 was digested with 5 μg/mL mocarhagin in TBS, 2 mM calcium chloride; 1 mg/mL BSA for 20 min at 37C. The ability of sPSGL-1.T7 to bind P-selectin was assessed by precipitation with the P-selectin IgG chimera LECγ1 (Sako et al.) preabsorbed onto protein A Sepharose beads in TBS, 2 mM calcium chloride, 1 mg/mL BSA for 4 h at 4C. A control experiment was also performed where the LECγ1 protein A Sepharose beads were pre-treated with mocarhagin and then exhaustively washed prior to presentation of sPSGL-1.T7. For immunoprecipitation analysis of untreated and mocarhagin treated sPSGL-1.T7, the protease was inactivated by the addition of 5 mM EDTA. sPSGL-1.T7 was then immunoprecipitated with anti-PSGL-1 polyclonal antibodies Rb3026 (raised against COS produced sPSGL-1.T7; Sako et al.) or Rb3443 (raised against the N-terminal peptide of PACE cleaved PSGL-1:QATEYEYLDYDFLPE, SEQ ID NO:4).

Example 4

Peptide Cleavage Assay

A digestion buffer (10 mM MOPS, 150 mM NaCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 0.02% NaN 3 , pH 7.5) and a peptide substrate solution (pyroEATEYEYLDYDFLPE (SEQ ID NO:3), 10 mM in DMSO) were prepared. 2.5 μL peptide substrate solution (250 μM final substrate concentration) was combined with mocarhagin sample material (10 μg/mL final mocarhagin concentration) and adjusted to 100 μL with digestion buffer using no less than 75 μL. This mixture was digested at 37C for 16 hours in parallel with a control sample (no mocarhagin added).

50 μL aliquots of the digested samples were run on an RP-HPLC column (Vydac C18 218TP54, 4.6×250 mm), using the following solvents: solvent A, 0.1% TFA in H 2 O; solvent B, 0.075% TFA in 90% AcN; flow rate 1 mL/min. The presence of peptides in the eluate was measured by absorbance at 214 nm, 260 nm and 280 nm. A positive assay result was indicated by observing elution of two peptide peaks in the tested sample which both elute earlier than the single peptide peak observed in the negative control.

Example 5

Cloning of Polynucleotide Encoding Mocarhagin Protein

Venom glands from five Mozambiquan spitting cobras, Naja mossambica mossambica , were dissected at two hour intervals, two to twelve hours following stimulation of venom production. Poly A+RNA was isolated from total RNA of the pooled gland tissue using an Oligotex Direct mRNA kit (Qiagen, Chatsworth, Calif.). Synthesis of cDNA was performed using Superscript Choice System (Gibco BRL, Gaithersberg, Md.) using oligo dT and random hexamer primers, EcoRI adapters. The cDNA was ligated with EcoRI digested lambda Zap II cloning vector (Stratagene, La Jolla, Calif.).

Using the above cDNA preparation as template, a PCR reaction was performed using degenerate oligonucleotides based on the N-terminal 30 residue amino acid sequence described above. The sequences of the forward primer consisted of 5′- ACNCCNGARCARGAY (SEQ ID NO:19). The sequences of the reverse primer consisted of 5′-RTAYTTYCKRTACAT (SEQ ID NO:20). A resulting 84 bp product was subsequently identified and DNA sequencing confirmed the sequence encoded 30 amino acid residues having a high degree of homology to the previously determined amino acid sequence. Two oligonucleotides 24 nucleotides in length, 5′-CAGGACAGGTACTTGCAGGCCAAA (SEQ ID NO:21) and 5′-ATCGAGTTTTACGTGGTTGTGGAC (SEQ ID NO:22), were synthesized based on the PCR product sequence and used as 32 p hybridization probes to screen approximately 10 6 plaques of plated lambda Zap II library. Duplicate sets of Duralose filters (Stratagene, La Jolla, Calif.) were hybridized seperately with each 32 p hybridization probe in 5×SSC, 5×Denhardt's, 0.1% SDS, 50 ug/mi yeast tRNA 16 hrs @40C. Filters were washed with 4×SSC, 0.1% SDS @ room temperature, then twice at 45C for 30 min. Autoradiography was −70C overnight with intensifying screen. Plaques showing positive hybridization to both probes were isolated and ultimately characterized by nucleotide sequencing.

Clones NMM-1, NMM-2, NMM-3, NMM-9, NMM-10, NMM-12 and NMM-13, described above, were isolated by this technique.

Example 6

Enterokinase Cleavage of NMM-9ek

COS cells were transfected with plasmid pED.NMM9ek, which included the cDNA sequence of SEQ ID NO:17 as an insert. This construct contains a novel enterokinase cleavage site between the propeptide and mature peptide of mocarhagin. After 48 hours, the transfected cells were washed in serum free medium, labelled with 35 S methionine for 6 hours, and the serum free conditioned mediuma was harvested. Purified bovine enterokinase (La Vallie et al., 1993, J. BIOL. Chem. 268:23311-23317) was added at various concentrations to 100 ul conditioned medium with 10 mM Tris pH8 and 1 mM CaCl 2 , and incubate at 37C overnight. Soy Trypsin Inhibitor resin was added to remove the enterokinase from the reaction mixture. The resin was pelleted by centrifugation and the supernatant was then immunoprecipitated with rabbit polyclonal antibodies raised against mocarhagin purified from cobra venom.

Following SDS-PAGE and autoradiography, a novel ˜50 kD band appeared in the sample lane where 50 nanograms of purified bovine enterokinase had been inclubated with the conditioned medium (see FIG. 3 ). This band is consistent with the expected molecular weight of the mature protease when the propeptide (˜23 kD) is cleaved off.

22 30 amino acids amino acid single linear peptide NO 1Thr Asn Thr Pro Glu Gln Asp Arg Tyr Leu Gln Ala Lys Lys Tyr Ile1 5 10 15Glu Phe Tyr Val Val Val Asp Asn Val Met Tyr Arg Lys Tyr 20 25 30 48 amino acids amino acid single linear peptide NO 2Thr Asn Thr Pro Glu Gln Asp Arg Tyr Leu Gln Ala Lys Lys Tyr Ile1 5 10 15Glu Phe Tyr Val Val Val Asp Asn Val Met Tyr Arg Lys Tyr Thr Gly 20 25 30Lys Leu His Val Ile Thr Xaa Xaa Val Tyr Glu Met Asn Ala Leu Asn 35 40 45 15 amino acids amino acid single linear peptide 3Glu Ala Thr Glu Tyr Glu Tyr Leu Asp Tyr Asp Phe Leu Pro Glu1 5 10 15 15 amino acids amino acid single linear peptide 4Gln Ala Thr Glu Tyr Glu Tyr Leu Asp Tyr Asp Phe Leu Pro Glu1 5 10 15 2050 base pairs nucleic acid double linear cDNA CDS 78..1940 5AGTCAATAGG AGAAGAGCTC AGGTTGGCTT GGAAGCAGAA AGAGATTCCT GTCCACCACT 60CCAATCCAGG CTCCAAAATG ATCCAAGCTC TCTTGGTAGC TATATGCTTA GCGGTTTTTC 120CATATCAAGG GAGCTCTATA ATCCTGGAAT CCGGGAATGT TAATGATTAT GAAGTAGTGT 180ATCCACAAAA AGTGCCTGCA TTGTCCAAAG GAGGAGTTCA GAATCCTCAG CCAGAGACCA 240AGTATGAAGA TACAATGCAA TATGAATTTC ACGTGAACGG AGAGCCAGTG GTCCTTCACT 300TAGAAAGAAA TAAAGGACTT TTTTCAGAAG ATTACACTGA AACTCATTAT GCCCCTGATG 360GCAGAGAAAT TACAACAAGC TCTCCAGTTC AGGATCACTG CTATTATCAT GGTTACATTC 420AGAATGAAGC TGACTCAAGT GCAGTCATCA GTGCATGTGA TGGCTTGAAA GGACATTTCA 480AGCATCAAGG GGAGACATAC TTTATTGAGC CCTTGGAGCT TTCTGACAGT GAAGCCCATG 540CAATATACAA AGATGAAAAT GTAGAAGAAG AGGAAGAGAT CCCCAAAATC TGTGGGGTTA 600CCCAGACTAC TTGGGAATCA GATGAGCCGA TTGAAAAGTC CTCTCAGTTA ACTAATACTC 660CTGAACAAGA CAGGTACTTG CAGGCCAAAA AATACATCGA GTTTTACGTG GTTGTGGACA 720ATGTAATGTA CMGRAAATAC ACCGGCAAGT TACATGTTAT AACAAGAAGA GTATATGAAA 780TGGTCAACGC TTTAAATACG ATGTACAGAC GTTTGAATTT TCACATAGCA CTGATTGGCC 840TAGAAATTTG GTCCAACGGA AATGAGATTA ATGTGCAATC AGACGTGCAG GCCACTTTGG 900ACTTATTTGG AGAATGGAGA GAAAATAAAT TGCTGCCACG CAAAAGGAAT GATAATGCTC 960AGTTACTCAC GAGCACTGAG TTCAATGGAA CTACTACAGG ACTTGGTTAC ATAGGCTCCC 1020TCTGTAGTCC GAAGAAATCT GTGGCAGTTG TTCAGGATCA TAGCAAAAGC ACAAGCATGG 1080TGGCAATTAC AATGGCCCAT CAGATGGGTC ATAATCTGGG CATGAATGAT GACAGAGCTT 1140CCTGTACTTG TGGTTCTAAC AAATGCATTA TGTCTACAAA ATATTATGAA TCTCTTTCTG 1200AGTTCAGCTC TTGTAGTGTC CAGGAACATC GGGAGTATCT TCTTAGAGAC AGACCACAAT 1260GCATTCTCAA CAAACCCTCG CGCAAAGCTA TTGTTACACC TCCAGTTTGT GGAAATTACT 1320TTGTGGAGCG GGGAGAAGAA TGTGACTGTG GCTCTCCTGA GGATTGTCAA AATACCTGCT 1380GTGATGCTGC AACTTGTAAA CTGCAACATG AGGCACAGTG TGACTCTGGA GAGTGTTGTG 1440AGAAATGCAA ATTTAAGGGA GCAGGAGCAG AATGCCGGGC AGCAAAGAAT GACTGTGACT 1500TTCCTGAACT CTGCACTGGC CGATCTGCTA AGTGTCCCAA GGACAGCTTC CAGAGGAATG 1560GACATCCATG CCAAAACAAC CAAGGTTACT GCTACAATGG GACATGTCCC ACCTTGACAA 1620ACCAATGTGC TACTCTCTGG GGGCCAGGTG CAAAAATGTC TCCAGGTTTA TGTTTTATGT 1680TGAACTGGAA TGCCCGAAGT TGTGGCTTGT GCAGAAAGGA AAATGGCAGA AAGATTCTAT 1740GTGCAGCAAA GGATGTAAAG TGTGGCAGGT TATTTTGCAA AAAGAAAAAC TCGATGATAT 1800GCCACTGCCC ACTCCATCAA AGGACCCAAA TTATGGAATG GTTGCACCTG GAACAAAATG 1860TGGAGTTAAA AAGGTGTGCA GAAACAGGCA ATGTGTTAAA GTATAGACAG CCAACTGATC 1920AAGCACTGCT TCTCTCAATT TGATTTTGGA GATCCTCCTT CCAGAAGGCT TTCCTCAAGT 1980CCAAAGAGAC CCATCTGTCT TTATCCTACT AGTAAATCAC TCTTAGCTTT CAAAAAAAAA 2040AAAAGTCGAC 2050 621 amino acids amino acid linear protein 6Met Ile Gln Ala Leu Leu Val Ala Ile Cys Leu Ala Val Phe Pro Tyr1 5 10 15Gln Gly Ser Ser Ile Ile Leu Glu Ser Gly Asn Val Asn Asp Tyr Glu 20 25 30Val Val Tyr Pro Gln Lys Val Pro Ala Leu Ser Lys Gly Gly Val Gln 35 40 45Asn Pro Gln Pro Glu Thr Lys Tyr Glu Asp Thr Met Gln Tyr Glu Phe 50 55 60His Val Asn Gly Glu Pro Val Val Leu His Leu Glu Arg Asn Lys Gly65 70 75 80Leu Phe Ser Glu Asp Tyr Thr Glu Thr His Tyr Ala Pro Asp Gly Arg 85 90 95Glu Ile Thr Thr Ser Ser Pro Val Gln Asp His Cys Tyr Tyr His Gly 100 105 110Tyr Ile Gln Asn Glu Ala Asp Ser Ser Ala Val Ile Ser Ala Cys Asp 115 120 125Gly Leu Lys Gly His Phe Lys His Gln Gly Glu Thr Tyr Phe Ile Glu 130 135 140Pro Leu Glu Leu Ser Asp Ser Glu Ala His Ala Ile Tyr Lys Asp Glu145 150 155 160Asn Val Glu Glu Glu Glu Glu Ile Pro Lys Ile Cys Gly Val Thr Gln 165 170 175Thr Thr Trp Glu Ser Asp Glu Pro Ile Glu Lys Ser Ser Gln Leu Thr 180 185 190Asn Thr Pro Glu Gln Asp Arg Tyr Leu Gln Ala Lys Lys Tyr Ile Glu 195 200 205Phe Tyr Val Val Val Asp Asn Val Met Tyr Arg Lys Tyr Thr Gly Lys 210 215 220Leu His Val Ile Thr Arg Arg Val Tyr Glu Met Val Asn Ala Leu Asn225 230 235 240Thr Met Tyr Arg Arg Leu Asn Phe His Ile Ala Leu Ile Gly Leu Glu 245 250 255Ile Trp Ser Asn Gly Asn Glu Ile Asn Val Gln Ser Asp Val Gln Ala 260 265 270Thr Leu Asp Leu Phe Gly Glu Trp Arg Glu Asn Lys Leu Leu Pro Arg 275 280 285Lys Arg Asn Asp Asn Ala Gln Leu Leu Thr Ser Thr Glu Phe Asn Gly 290 295 300Thr Thr Thr Gly Leu Gly Tyr Ile Gly Ser Leu Cys Ser Pro Lys Lys305 310 315 320Ser Val Ala Val Val Gln Asp His Ser Lys Ser Thr Ser Met Val Ala 325 330 335Ile Thr Met Ala His Gln Met Gly His Asn Leu Gly Met Asn Asp Asp 340 345 350Arg Ala Ser Cys Thr Cys Gly Ser Asn Lys Cys Ile Met Ser Thr Lys 355 360 365Tyr Tyr Glu Ser Leu Ser Glu Phe Ser Ser Cys Ser Val Gln Glu His 370 375 380Arg Glu Tyr Leu Leu Arg Asp Arg Pro Gln Cys Ile Leu Asn Lys Pro385 390 395 400Ser Arg Lys Ala Ile Val Thr Pro Pro Val Cys Gly Asn Tyr Phe Val 405 410 415Glu Arg Gly Glu Glu Cys Asp Cys Gly Ser Pro Glu Asp Cys Gln Asn 420 425 430Thr Cys Cys Asp Ala Ala Thr Cys Lys Leu Gln His Glu Ala Gln Cys 435 440 445Asp Ser Gly Glu Cys Cys Glu Lys Cys Lys Phe Lys Gly Ala Gly Ala 450 455 460Glu Cys Arg Ala Ala Lys Asn Asp Cys Asp Phe Pro Glu Leu Cys Thr465 470 475 480Gly Arg Ser Ala Lys Cys Pro Lys Asp Ser Phe Gln Arg Asn Gly His 485 490 495Pro Cys Gln Asn Asn Gln Gly Tyr Cys Tyr Asn Gly Thr Cys Pro Thr 500 505 510Leu Thr Asn Gln Cys Ala Thr Leu Trp Gly Pro Gly Ala Lys Met Ser 515 520 525Pro Gly Leu Cys Phe Met Leu Asn Trp Asn Ala Arg Ser Cys Gly Leu 530 535 540Cys Arg Lys Glu Asn Gly Arg Lys Ile Leu Cys Ala Ala Lys Asp Val545 550 555 560Lys Cys Gly Arg Leu Phe Cys Lys Lys Lys Asn Ser Met Ile Cys His 565 570 575Cys Pro Leu His Gln Arg Thr Gln Ile Met Glu Trp Leu His Leu Glu 580 585 590Gln Asn Val Glu Leu Lys Arg Cys Ala Glu Thr Gly Asn Val Leu Lys 595 600 605Tyr Arg Gln Pro Thr Asp Gln Ala Leu Leu Leu Ser Ile 610 615 620 2297 base pairs nucleic acid double linear cDNA 7GTCGACCAGT CAACAGGAGA AAAGCTCAGG TTGGCTTGGA AGCAGAAAGA GATTCCTGTC 60CACCAGTCCA ATCCAGGCTC CAAAATGATC CAAGCTCTCT TGGTAATTAT ATGCTTAGCG 120GTTTTTCCAT ATCAAGGGAG CTCTATAATC CTGGAATCTG GGAATGTTAA TGATTATGAA 180GTTGTGTATC CACAAAAAGT GCCTGCATTG CTCAAAGGAG GAGTTCAGAA TCCTCAGCCA 240GAGACCAAGT ATGAAGATAC AATGCAATAT GAATTTCAAG TGAATGGAGA GCCAGTAGTC 300CTTCACTTAG AAAGAAATAA AGGACTTTTT TCAGAAGATT ACACTGAAAC TCATTATGCC 360CCTGATGGCA GAGAAATTAC AACAAGCCCT CCGGTTCAGG ATCACTGCTA TTATCATGGT 420TACATTCAGA ATGAAGCTGA CTCAAGTGCA ATCATCAGTG CATGTGATGG CTTGAAAGGA 480CATTTCAAGC ATCAAGGGGA GACATACTTT ATTGAGCCCT TGAAGCTTTT CGACAGTGAA 540TCTCATGCAA TCTACAAAGA TGAAAATGTA GAAAACGAGG ATGAGACCCC CGAAACCTGT 600GGGGTAACCG AGACTACTTG GGAGTCAGAT GAGTCCATCG AAAAGACCTC TCAGTTAACT 660AACACTCCTG AACAAGACGG GTACTTGCAG GCCAAAAAAT ACATCGAGTT TTACGTGGTT 720GTGGACAACA GAATGTACAG GTATTACAAA CGCAATGAAC CTGCTATAAA AAGAAGAGTA 780TATGAAATGG TCAACGCTGT AAATACGAAG TACAGACCTT TGAAAATTCA CATAACACTG 840ATTGGCCTAG AAATTTGGTC CAACCATGAT AAGTTTGAAG TGAAGCCAGT AGCGGGTGCC 900ACTTTGAAAT CATTTCGAGA TTGGAGAGAA ACAGTTTTGC TGCCACGCAA AAGGAATGAT 960AACGCTCAGT TACTCACGGG CATTGACTTC AATGGAACTG TTGTGGGAAT TGCTTACACG 1020GGCACCCTCT GCACTCAGAA TTCTGTAGCA GTTGTTCAGG ATTATAACCG AAAAATAAGC 1080ATGGTGGCAT CTACAATGGC CCATGAGTTG GGTCATAATC TGGGCCTTCA TCATGACGGA 1140GCTTCCTGTA TTTGCAGTCT TAGACCATGC ATTATGTCTA AGGGACGGAC TGCACCTGCC 1200TTTCAGTTCA GCTCTTGTAG TGTCCGGGAG TATCGGGAGT ATCTTCTTAG AGAAAGACCA 1260CAATGCATTC TCAACAAACC CTTGAGCACA GATACTGTTT CACCTGCAAT TTGTGGAAAT 1320TACTTTGTGG AGGAGGGAGA AGAATGTGAC TGTGGCTCTC CTGCGGATTG TCAAAGTGCC 1380TGCTGCGATG CTGCAACTTG TTAGTTTAAG GGAGAAGAAG CAGAATGCCG GGCAGCAAAG 1440GATGACTGTG ACTTGCCTGA ACTCTGCACT GGCCGATCTG TGGAGTGTCC CACGGACAGC 1500TTGCAGAAGA ATGGACATCC ATGTCAAAAC AACAAAGGTT ACTGCTACAA TGGGGCATGT 1560CCCACCTTCA CAAACCAATG TATTGCTCTC ATGGGGACAG ATTTTACTGT GAGTCCAGAT 1620GGATGTTTTG ACTTGAACGT GAGAGGGAAT GATGTAAGCC ACTGCAGAAA GGAAAATGGT 1680GCAAAGATTC CATGTGCAGC AAAGGATGTA AAGTGTGGCA GGTTATACTG CACAGAGAGA 1740GACACAATGT CATGCCGATT CCCACTGGAC CCAGATGGTG TAATGGCTGA ACCTGGAACA 1800AAATGTGGAG ATGGAATGGT GTGCAGCAAC GGTCAGTGTG TTAATGTGCA GACAGCCTAC 1860TGATCAAGCA CTGGCTTCTC TCAATTTGAT TTTGGAGATC CTCCTTCCAG AACGCTTCCC 1920TCAAGTCCAA AGAGACCCAT CTGTCTTTAT CCTACTAGTA AATCACTCTT AGCTTTCAGA 1980TGGTATCTAA AATTTATAAT ATTTCTTCTC CATAATTTAA ACTGGTAATC TTTTGCTAAA 2040ATCAGACCTT TTCCCTGCCA CAAAGCTCCA TGGTCATGTA CAGCACCAAA GGCTTATTTG 2100CGAATAAGAA AAAAAAATGG CAATTTTACA GTTTCCCAAT TGCAATGCAT ATTGAATGCA 2160ACAAGCTCTG CCCTTTGAGC TGGCGTATTC AAAGGCAATG CTCCCTCTCC CAAAATTATA 2220CGCTGGCTTT CCAAGATGTA GCTGCTTCCA TCAATAAACT ATTCTCATTC TGCAAAAAAA 2280AAAAAAAAAA AGTCGAC 2297 439 amino acids amino acid linear protein 8Met Ile Gln Ala Leu Leu Val Ile Ile Cys Leu Ala Val Phe Pro Tyr1 5 10 15Gln Gly Ser Ser Ile Ile Leu Glu Ser Gly Asn Val Asn Asp Tyr Glu 20 25 30Val Val Tyr Pro Gln Lys Val Pro Ala Leu Leu Lys Gly Gly Val Gln 35 40 45Asn Pro Gln Pro Glu Thr Lys Tyr Glu Asp Thr Met Gln Tyr Glu Phe 50 55 60Gln Val Asn Gly Glu Pro Val Val Leu His Leu Glu Arg Asn Lys Gly65 70 75 80Leu Phe Ser Glu Asp Tyr Thr Glu Thr His Tyr Ala Pro Asp Gly Arg 85 90 95Glu Ile Thr Thr Ser Pro Pro Val Gln Asp His Cys Tyr Tyr His Gly 100 105 110Tyr Ile Gln Asn Glu Ala Asp Ser Ser Ala Ile Ile Ser Ala Cys Asp 115 120 125Gly Leu Lys Gly His Phe Lys His Gln Gly Glu Thr Tyr Phe Ile Glu 130 135 140Pro Leu Lys Leu Phe Asp Ser Glu Ser His Ala Ile Tyr Lys Asp Glu145 150 155 160Asn Val Glu Asn Glu Asp Glu Thr Pro Glu Thr Cys Gly Val Thr Glu 165 170 175Thr Thr Trp Glu Ser Asp Glu Ser Ile Glu Lys Thr Ser Gln Leu Thr 180 185 190Asn Thr Pro Glu Gln Asp Gly Tyr Leu Gln Ala Lys Lys Tyr Ile Glu 195 200 205Phe Tyr Val Val Val Asp Asn Arg Met Tyr Arg Tyr Tyr Lys Arg Asn 210 215 220Glu Pro Ala Ile Lys Arg Arg Val Tyr Glu Met Val Asn Ala Val Asn225 230 235 240Thr Lys Tyr Arg Pro Leu Lys Ile His Ile Thr Leu Ile Gly Leu Glu 245 250 255Ile Trp Ser Asn His Asp Lys Phe Glu Val Lys Pro Val Ala Gly Ala 260 265 270Thr Leu Lys Ser Phe Arg Asp Trp Arg Glu Thr Val Leu Leu Pro Arg 275 280 285Lys Arg Asn Asp Asn Ala Gln Leu Leu Thr Gly Ile Asp Phe Asn Gly 290 295 300Thr Val Val Gly Ile Ala Tyr Thr Gly Thr Leu Cys Thr Gln Asn Ser305 310 315 320Val Ala Val Val Gln Asp Tyr Asn Arg Lys Ile Ser Met Val Ala Ser 325 330 335Thr Met Ala His Glu Leu Gly His Asn Leu Gly Leu His His Asp Gly 340 345 350Ala Ser Cys Ile Cys Ser Leu Arg Pro Cys Ile Met Ser Lys Gly Arg 355 360 365Thr Ala Pro Ala Phe Gln Phe Ser Ser Cys Ser Val Arg Glu Tyr Arg 370 375 380Glu Tyr Leu Leu Arg Glu Arg Pro Gln Cys Ile Leu Asn Lys Pro Leu385 390 395 400Ser Thr Asp Thr Val Ser Pro Ala Ile Cys Gly Asn Tyr Phe Val Glu 405 410 415Glu Gly Glu Glu Cys Asp Cys Gly Ser Pro Ala Asp Cys Gln Ser Ala 420 425 430Cys Cys Asp Ala Ala Thr Cys 435 2335 base pairs nucleic acid double linear cDNA 9GTCGACCTCA GGTTGGCTTG GAAGCAGAAA GAGATTCCTA TCCACCACTC CAATCCAGGC 60TCCAAAATGA TCCAAGCTCT CTTGGTAGCT ATATGCTTAG CGGTTTTTCC ATATCAAGGG 120AGCTCTATAA TCCTGGAATC CGGGAATGTT AATGATTATG AAGTAGTGTA TCCACAAAAA 180GTGCCTGCAT TGTCCAAAGG AGGAGTTCAG AATCCTCAGC CAGAGACCAA GTATGAAGAT 240ACAATGCAAT ATGAATTTCA AGTGAATGGA GAGCCAGTAG TCCTTCACCT AGAAAGAAAT 300AAAGGACTTT TTTCAGAAGA TTACACTGAA ACTCATTATG CCTCTGATGG CAGAGAAATT 360ACAACAAGCC CACTCGTTCA GGATCACTGC TATTATCATG GTTACATTCA GAATGAAGCT 420GACTCAAGTG CAGTCATCAG TGCATGCGAT GGCTTGAAAG GACATTTCGA GCTTCAAGGG 480GAGACATACT TTATTGAACC CTTGAAGATT TCCGACAGTG AAGCCCATGC AATCTACAAA 540GATGAAAATG TAGAAAACGA GGATGAGACC CCCGAAACCT GTGGGGTAAC CGAGACTACT 600TGGGAGTCAG ATGAGTCCAT TGAAAAGACC TCTCAGTTAA CTAACACTCC TGAACAAGAC 660AGGTACTTGC AGGCCAAAAA ATACCTCGAG TTTTACGTGG TTGTGGACAA CATAATGTAC 720AGGCATTACA AACGCGATAA ACCTGTTATA AAAAGAAGAG TATATGAAAT GATCAACACT 780ATGAATATGG TGTACAATCG TTTGAATTTT CACATAGCAC TGATTGGCCT AGAAATTTGG 840TCCAACAGAA ATGAGATTAA TGTGCAATCA GACGTGCAGG CCACTTTGGA CTTATTTGGA 900GAATGGAGAG AAAAAAAATT GCTGCCACGC AAAAGGAATG ATAATGCTCA GTTACTCACG 960GGTATTGACT TCAAAGGAAC TCCTGTAGGA CTTGCTTACA TAGGTTCCAT CTGCAATCCG 1020AAGAGTTCTG TAGCAGTTGT TCAGGATTAT AGCAGTAGAA CAAGCATGGT GGCAATTACA 1080ATGGCCCATG AGATGGGTCA TAATATGGGC ATTCATCATG ACGGACCTTC CTGTACTTGT 1140GGTTCTAACA AATGCGTTAT GTCTACAAGA CGTACTGAAC CTGCCTATCA GTTCAGCTCT 1200TGTAGTGTCC GGGAACATCA GGAGTATCTT CTTAGAGACA GACCACAATG CATTCTCAAC 1260AAACCCTTGA GCACAGATAT TGTTTCACCT CCAATTTGTG GAAATAACTT TGTGGAGGTG 1320GGAGAAGAAT GTGACTGTGG CTCTCCTGCG GATTGTCAAA GTGCCTGCTG CGACGCTACA 1380ACTTGTAAAC TACAACCTCA TGCACAGTGT GACTCCGAAG GGTGTTGTGA GAAATGCAAA 1440TTTAAGGGAG CAGGAGCAGA ATGCCGGGCA GCAAAGGATG ACTGTGACTT GCCTGAACTC 1500TGCACTGGCC AATCTGCTGA GTGTCCCACA GACATCTTCC AGAGGAATGG ACTTCCATGC 1560CAAAACAACG AAGGTTACTG CTACAATGGG AAATGCCCCA TCATGACAAA CCAATGTATT 1620GCTCTCCGGG GACCAGGTGT AAAAGTATCT CGAGATAGCT GTTTTACATT GAACCAGAGA 1680ACCAGTGGTT GTGGCTTGTG CAGAATGGAA TATGGTAGAA AGATTCCATG TGCAGCAAAG 1740GATGTAAAGT GTGGCAGGTT ATTTTGCAAA AAGGGAAACT CGATGATATG CAACTGCTCA 1800GTTTCACCAC GTGACCCAAG TTATGGAATG GTTGAACCTG GAACAAAATG TGGAGATGGA 1860ATGGTGTGCA GCAACAGGCA GTGTGTTGAT GTGAAGACAG CCTACTGATC AAGCACTGGC 1920TTCTCTCAAT TTGATTTTGG AGGTCCTCCT TCCAGAACGC TTCCCTCAAG TCCAAAGAGA 1980CCCATCTGTC TTTATCCTAC TAGTAAATCA CTCTTAGCTT TCAGATGGTA TCTAAAATTT 2040AAAATATTTC TTCTCCATAA TTTAAACTGG TAATCTTTTG CTAAAATCAG ACCTTTTCCC 2100TGCCACAAAG CTCCATGGTC ATGTACAGCA CCAAAGGCTT ATTTGCTAAC AAGAAAAAAA 2160ATGGCCATTT TACTGTTTGC CAATTGCAAT TCACATTTAA TGCAACAAGC TCTGCCCTTT 2220GAGCTGGCGT ACTCAAAGGC AATGCTCCCT CTCCCAAAAT TATACGCTGG CTTTCCAAGA 2280TGTAGCTGCT TCCATCAATA AACTATTCTC ATTCTGAAAA AAAAAAAAAG TCGAC 2335 613 amino acids amino acid linear protein 10Met Ile Gln Ala Leu Leu Val Ala Ile Cys Leu Ala Val Phe Pro Tyr1 5 10 15Gln Gly Ser Ser Ile Ile Leu Glu Ser Gly Asn Val Asn Asp Tyr Glu 20 25 30Val Val Tyr Pro Gln Lys Val Pro Ala Leu Ser Lys Gly Gly Val Gln 35 40 45Asn Pro Gln Pro Glu Thr Lys Tyr Glu Asp Thr Met Gln Tyr Glu Phe 50 55 60Gln Val Asn Gly Glu Pro Val Val Leu His Leu Glu Arg Asn Lys Gly65 70 75 80Leu Phe Ser Glu Asp Tyr Thr Glu Thr His Tyr Ala Ser Asp Gly Arg 85 90 95Glu Ile Thr Thr Ser Pro Leu Val Gln Asp His Cys Tyr Tyr His Gly 100 105 110Tyr Ile Gln Asn Glu Ala Asp Ser Ser Ala Val Ile Ser Ala Cys Asp 115 120 125Gly Leu Lys Gly His Phe Glu Leu Gln Gly Glu Thr Tyr Phe Ile Glu 130 135 140Pro Leu Lys Ile Ser Asp Ser Glu Ala His Ala Ile Tyr Lys Asp Glu145 150 155 160Asn Val Glu Asn Glu Asp Glu Thr Pro Glu Thr Cys Gly Val Thr Glu 165 170 175Thr Thr Trp Glu Ser Asp Glu Ser Ile Glu Lys Thr Ser Gln Leu Thr 180 185 190Asn Thr Pro Glu Gln Asp Arg Tyr Leu Gln Ala Lys Lys Tyr Leu Glu 195 200 205Phe Tyr Val Val Val Asp Asn Ile Met Tyr Arg His Tyr Lys Arg Asp 210 215 220Lys Pro Val Ile Lys Arg Arg Val Tyr Glu Met Ile Asn Thr Met Asn225 230 235 240Met Val Tyr Asn Arg Leu Asn Phe His Ile Ala Leu Ile Gly Leu Glu 245 250 255Ile Trp Ser Asn Arg Asn Glu Ile Asn Val Gln Ser Asp Val Gln Ala 260 265 270Thr Leu Asp Leu Phe Gly Glu Trp Arg Glu Lys Lys Leu Leu Pro Arg 275 280 285Lys Arg Asn Asp Asn Ala Gln Leu Leu Thr Gly Ile Asp Phe Lys Gly 290 295 300Thr Pro Val Gly Leu Ala Tyr Ile Gly Ser Ile Cys Asn Pro Lys Ser305 310 315 320Ser Val Ala Val Val Gln Asp Tyr Ser Ser Arg Thr Ser Met Val Ala 325 330 335Ile Thr Met Ala His Glu Met Gly His Asn Met Gly Ile His His Asp 340 345 350Gly Pro Ser Cys Thr Cys Gly Ser Asn Lys Cys Val Met Ser Thr Arg 355 360 365Arg Thr Glu Pro Ala Tyr Gln Phe Ser Ser Cys Ser Val Arg Glu His 370 375 380Gln Glu Tyr Leu Leu Arg Asp Arg Pro Gln Cys Ile Leu Asn Lys Pro385 390 395 400Leu Ser Thr Asp Ile Val Ser Pro Pro Ile Cys Gly Asn Asn Phe Val 405 410 415Glu Val Gly Glu Glu Cys Asp Cys Gly Ser Pro Ala Asp Cys Gln Ser 420 425 430Ala Cys Cys Asp Ala Thr Thr Cys Lys Leu Gln Pro His Ala Gln Cys 435 440 445Asp Ser Glu Gly Cys Cys Glu Lys Cys Lys Phe Lys Gly Ala Gly Ala 450 455 460Glu Cys Arg Ala Ala Lys Asp Asp Cys Asp Leu Pro Glu Leu Cys Thr465 470 475 480Gly Gln Ser Ala Glu Cys Pro Thr Asp Ile Phe Gln Arg Asn Gly Leu 485 490 495Pro Cys Gln Asn Asn Glu Gly Tyr Cys Tyr Asn Gly Lys Cys Pro Ile 500 505 510Met Thr Asn Gln Cys Ile Ala Leu Arg Gly Pro Gly Val Lys Val Ser 515 520 525Arg Asp Ser Cys Phe Thr Leu Asn Gln Arg Thr Ser Gly Cys Gly Leu 530 535 540Cys Arg Met Glu Tyr Gly Arg Lys Ile Pro Cys Ala Ala Lys Asp Val545 550 555 560Lys Cys Gly Arg Leu Phe Cys Lys Lys Gly Asn Ser Met Ile Cys Asn 565 570 575Cys Ser Val Ser Pro Arg Asp Pro Ser Tyr Gly Met Val Glu Pro Gly 580 585 590Thr Lys Cys Gly Asp Gly Met Val Cys Ser Asn Arg Gln Cys Val Asp 595 600 605Val Lys Thr Ala Tyr 610 2288 base pairs nucleic acid double linear cDNA 11AGTCAACAGG AGAAAAGCTC AGGTTGGCTT GGAAGCAGAA AGAGATTCCT GTCCACCAGT 60CCAATCCAGG CTCCAAAATG ATCCAAGCTC TCTTGGTAAT TATATGCTTA GTGGTTTTTC 120CATATCAAGG GAGCTCTATA ATCCTGGAAT CTGGGAATGT TAATGATTAT GAAGTTGTGT 180ATCCACAAAA AGTGCCTGCA TTGCTCAAAG GAGGAGTTCA GAATCCTCAG CCAGAGACCA 240AGTATGAAGA TACAATGCAA TATGAATTTC AAGTGAATGG AGAGCCAGTA GTCCTTCACT 300TAGAAAGAAA TAAAGGACTT TTTTCAGAAG ATTACACTGA AACTCATTAT GCCCCTGATG 360GCAGAGAAAT TACAACAAGC CCTCCGGTTC AGGATCACTG CTATTATCAT GGTTACATTC 420AGAATGAAGC TGACTCAAGT GCAATCATCA GTGCATGTGA TGGCTTGAAA GGACATTTCA 480AGCATCAAGG GGAGACATAC TTTATTGAGC CCTTGAAGCT TTTCGACAGT GAATCCCATG 540CAATCTACAA AGATGAAAAT GTAGAAAACG AGGATGAGAC CCCCGAAACC TGTGGGGTAA 600CCGAGACTAC TTGGGAGTCA GATGAGTCCA TCGAAAAGAC CTCTCAGTTA ACTAACACTC 660CTGAACAAGA CGGGTACTTG CAGGCCAAAA AATACATCGA GTTTTACGTG GTTGTGGACA 720ACAGAATGTA CAGGTATTAC AAACGCAATG AACCTGCTAT AAAAAGAAGA GTATATGAAA 780TGGTCAACGC TGTAAATACG TACAGACCTT TGAAAATTCA CATAACACTG ATTGGCCTAG 840AAATTTGGTC CAACGATGAT AAGTTTGAAG TGAAGCCAGT AGCGGGTGCC ACTTTGAAAT 900CATTTCGAGA TTGGAGAGAA ACAGTTTTGC TGCCACGCAA AAGGAATGAT AACGCTCAGT 960TACTCACGGG CATTGACTTC AATGGAACTG TTGTGGGAAT TGCTTACACG GGCACCCTCT 1020GCACTCAGAA TTCTGTAGCA GTTGTTCAGG ATTATAACCG AAAAATAAGC ATGGTGGCAT 1080CTACAATGGC CCATGAGTTG GGTCATAATC TGGGCCTTCA TCATGACGGA GCTTCCTGTA 1140TTTGCAGTCT TAGACCATGC ATTATGTCTA AGGGACGGAC TGCACCTGCC TTTCAGTTCA 1200GCTCTTGTAG TGTCCGGGAG TATCGGGAGT ATCTTCTTAG AGAAAGACCA CAATGCATTC 1260TCAACAAACC CTTGAGCACA GATACTGTTT CACCTGCAAT TTGTGGAAAT TACTTTGTGG 1320AGGAGGGAGA AGAATGTGAC TGTGGCTCTC CTGCGGATTG TCAAAGTGCC TGCTGCGATG 1380CTGCAACTTG TAAGTTTAAG GGAGAAGAAG CAGAATGCCG GGCAGCAAAG GATGACTGTG 1440ACTTGCCTGA ACTCTGCACT GGCCGATCTG TGGAGTGTCC CACGGACAGC TTGCAGAGGA 1500ATGGACATCC ATGTCAAAAC AACAAAGGTT ACTGCTACAA TGGGGCATGT CCCACCTTCA 1560CAAACCAATG TATTGCTCTC ATGGGGACAG ATTTTACTGT GAGTCCAGAT GGATGTTTTG 1620ACTTGAACGT GAGAGGGAAT TGATGTAAGC CACTGCAGAA AGGAAAATGG TGCAAAGATT 1680CCATGTGCAG CAAAGGATGT AAAGTGTGGC AGATTATACT GCACAGAGAG AGACACAATG 1740TCATGCCGAT TCCCACTGGA CCCAGATGGT GTTAATGGCT GAACCTGGAA CAAAATGTGG 1800AGATGGAATG GTGTGCAGCA ACGGTCAGTG TGTTAATGTG CAGACAGCCT ACTGATCAAG 1860CACTGGCTTC TCTCAATTTG ATTTTGGAGA TCCTCCTTCC AGAACGCTTC CCTCAAGTCC 1920AAAGAGACCC ATCTGTCTTT ATCCTACTAG TAAATCACTC TTAGCTTTCA GATGGTATCT 1980AAAATTTATA ATATTTCTTC TCCATAATTT AAACTGGTAA TCTTTTGCTA AAATCAGACC 2040TTTTCCCTGC CACAAAGCTC CATGGTCATG TACAGCACCA AAGGCTTATT TGCGAATAAG 2100AAAAAAAAAT GGCAATTTTA CAGTTTCCCA ATTGCAATGC ATATTGAATG CAACAAGCTC 2160TGCCCTTTGA GCTGGCGTAT TCAAAGGCAA TGCTCCCTCT CCCAAAATTA TACGCTGGCT 2220TTCCAAGATG TAGCTGCTTC CATCAATAAA CTATTCTCAT TCTGAAAAAA AAAAAAAAAA 2280AAGTCGAC 2288 521 amino acids amino acid linear protein 12Met Ile Gln Ala Leu Leu Val Ile Ile Cys Leu Val Val Phe Pro Tyr1 5 10 15Gln Gly Ser Ser Ile Ile Leu Glu Ser Gly Asn Val Asn Asp Tyr Glu 20 25 30Val Val Tyr Pro Gln Lys Val Pro Ala Leu Leu Lys Gly Gly Val Gln 35 40 45Asn Pro Gln Pro Glu Thr Lys Tyr Glu Asp Thr Met Gln Tyr Glu Phe 50 55 60Gln Val Asn Gly Glu Pro Val Val Leu His Leu Glu Arg Asn Lys Gly65 70 75 80Leu Phe Ser Glu Asp Tyr Thr Glu Thr His Tyr Ala Pro Asp Gly Arg 85 90 95Glu Ile Thr Thr Ser Pro Pro Val Gln Asp His Cys Tyr Tyr His Gly 100 105 110Tyr Ile Gln Asn Glu Ala Asp Ser Ser Ala Ile Ile Ser Ala Cys Asp 115 120 125Gly Leu Lys Gly His Phe Lys His Gln Gly Glu Thr Tyr Phe Ile Glu 130 135 140Pro Leu Lys Leu Phe Asp Ser Glu Ser His Ala Ile Tyr Lys Asp Glu145 150 155 160Asn Val Glu Asn Glu Asp Glu Thr Pro Glu Thr Cys Gly Val Thr Glu 165 170 175Thr Thr Trp Glu Ser Asp Glu Ser Ile Glu Lys Thr Ser Gln Leu Thr 180 185 190Asn Thr Pro Glu Gln Asp Gly Tyr Leu Gln Ala Lys Lys Tyr Ile Glu 195 200 205Phe Tyr Val Val Val Asp Asn Arg Met Tyr Arg Tyr Tyr Lys Arg Asn 210 215 220Glu Pro Ala Ile Lys Arg Arg Val Tyr Glu Met Val Asn Ala Val Asn225 230 235 240Thr Tyr Arg Pro Leu Lys Ile His Ile Thr Leu Ile Gly Leu Glu Ile 245 250 255Trp Ser Asn Asp Asp Lys Phe Glu Val Lys Pro Val Ala Gly Ala Thr 260 265 270Leu Lys Ser Phe Arg Asp Trp Arg Glu Thr Val Leu Leu Pro Arg Lys 275 280 285Arg Asn Asp Asn Ala Gln Leu Leu Thr Gly Ile Asp Phe Asn Gly Thr 290 295 300Val Val Gly Ile Ala Tyr Thr Gly Thr Leu Cys Thr Gln Asn Ser Val305 310 315 320Ala Val Val Gln Asp Tyr Asn Arg Lys Ile Ser Met Val Ala Ser Thr 325 330 335Met Ala His Glu Leu Gly His Asn Leu Gly Leu His His Asp Gly Ala 340 345 350Ser Cys Ile Cys Ser Leu Arg Pro Cys Ile Met Ser Lys Gly Arg Thr 355 360 365Ala Pro Ala Phe Gln Phe Ser Ser Cys Ser Val Arg Glu Tyr Arg Glu 370 375 380Tyr Leu Leu Arg Glu Arg Pro Gln Cys Ile Leu Asn Lys Pro Leu Ser385 390 395 400Thr Asp Thr Val Ser Pro Ala Ile Cys Gly Asn Tyr Phe Val Glu Glu 405 410 415Gly Glu Glu Cys Asp Cys Gly Ser Pro Ala Asp Cys Gln Ser Ala Cys 420 425 430Cys Asp Ala Ala Thr Cys Lys Phe Lys Gly Glu Glu Ala Glu Cys Arg 435 440 445Ala Ala Lys Asp Asp Cys Asp Leu Pro Glu Leu Cys Thr Gly Arg Ser 450 455 460Val Glu Cys Pro Thr Asp Ser Leu Gln Arg Asn Gly His Pro Cys Gln465 470 475 480Asn Asn Lys Gly Tyr Cys Tyr Asn Gly Ala Cys Pro Thr Phe Thr Asn 485 490 495Gln Cys Ile Ala Leu Met Gly Thr Asp Phe Thr Val Ser Pro Asp Gly 500 505 510Cys Phe Asp Leu Asn Val Arg Gly Asn 515 520 2309 base pairs nucleic acid double linear cDNA 13GTCGACGTCA ACAGGAGAAA AGCTCAGGTT GGCTTGGAAG CAGAAAGAGA TTCCTGTCCA 60CCAGTCCAAT CCAGGCTCCA AAATGATCCA AGCTCTCTTG GTAATTATAT GCTTAGTGGT 120TTTTCCATAT CAAGGGAGCT CTATAATCCT GGAATCTGGG AATGTTAATG ATTATGAAGT 180TGTGTATCCA CAAAAAGTGC CTGCATTGCT CAAAGGAGGA GTTCAGAATC CTCAGCCAGA 240GACCAAGTAT GAAGATACAA TGCAATATGA ATTTCAAGTG AATGGAGAGC CAGTAGTCCT 300TCACTTAGAA AGAAATAAAG GACTTTTTTC AGAAGATTAC ACTGAAACTC ATTATGCCCC 360TGATGGCAGA GAAATTACAA CAAGCCCTCC GGTTCAGGAT CACTGCTATT ATCATGGTTA 420CATTCAGAAT GAAGCTGACT CAAGTGCAAT CATCAGTGCA TGTGATGGCT TGAAAGGACA 480TTTCAAGCAT CAAGGGGAGA CATACTTTAT TGAGCCCTTG AAGCTTTTCG ACAGTGAATC 540CCATGCAATC TACAAAGATG AAAATGTAGA AAACGAGGAT GAGACCCCCG AAACCTGTGG 600GGTAACCGAG ACTACTTGGG AGTCAGATGA GTCCATCGAA AAGACCTCTC AGTTAACTAA 660CACTCCTGAA CAAGACGGGT ACTTGCAGGC CAAAAAATAC ATCGAGTTTT ACGTGGTTGT 720GGACAACAGA ATGTACAGGT ATTACAAACG CAATGAACCT GCTATAAAAA GAAGAGTATA 780TGAAATGGTC AACGCTGTAA ATACGAAGTA CAGACCTTTG AAAATTCACA TAACACTGAT 840TGGCCTAGAA ATTTGGTCCA ACGATGATAA GTTTGAAGTG AAGCCAGTAG CGGGTGCCAC 900TTTGAAATCA TTTCGAGATT GGAGAGAAAC AGTTTTGCTG CCACGCAAAA GGAATGATAA 960CGCTCAGTTA CTCACGGGCA TTGACTTCAA TGGAACTGTT GTGGGAATTG CTTACACGGG 1020CACCCTCTGC ACTCAGAATT CTGTAGCAGT TGTTCAGGAT TATAACCGAA AAATAAGCAT 1080GGTGGCATCT ACAATGGCCC ATGAGTTGGG TCATAATCTG GGCCTTCATC ATGACGGAGC 1140TTCCTGTATT TGCAGTCTTA GACCATGCAT TATGTCTAAG GGACGGACTG CACCTGCCTT 1200TCAGTTCAGC TCTTGTAGTG TCCGGGAGTA TCGGGAGTAT CTTCTTAGAG AAAGACCACA 1260ATGCATTCTC AACAAACCCT TGAGCACAGA TACTGTTTCA CCTGCAATTT GTGGAAATTA 1320CTTTGTGGAG GAGGGAGAAG AATGTGACTG TGGCTCTCCT GCGGATTGTC AAAGTGCCTG 1380CTGCGATGCT GCAACTTGTA AGTTTAAGGG AGAAGAAGCA GAATGCCGGG CAGCAAAGGA 1440TGACTGTGAC TTGCCTGAAC TCTGCACTGG CCGATCTGTG GAGTGTCCCA CGGACAGCTT 1500GCAGAGGAAT GGACATCCAT GTCAAAACAA CAAAGGTTAC TGCTACAATG GGGCATGTCC 1560CACCTTCACA AACCAATGTA TTGCTCTCAT GGGGACAGAT TTTACTGTGA GTCCAGATGG 1620ATGTTTTGAC TTGAACGTGA GAGGGAATGA TGTAAGCCAC TGCAGAAAGG AAAATGGTGC 1680AAAGATTCCA TGTGCAGCAA AGGATGTAAA GTGTGGCAGG TTATACTGCA CAGAGAGAGA 1740CACAATGTCA TGCCGATTCC CACTGGACCC AGATGGTGTA ATGGCTGAAC CTGGAACAAA 1800ATGTGGAGAT GGAATGGTGT GCAGCAACGG TCAGTGTGTT AATGTGCAGA CAGCCTACTG 1860ATCAAGCACT GGCTTCTCTC AATTTGATTT TGGAGATCCT CCTTCCAGAA CGCTTCCCTC 1920AAGTCCAAAG AGACCCATCT GTCTTTATCC TACTAGTAAA TCACTCTTAG CTTTCAGATG 1980GTATCTAAAA TTTATAATAT TTCTTCTCCA TAATTTAAAC TGGTAATCTT TTGCTAAAAT 2040CAGACCTTTT CCCTGCCACA AAGCTCCATG GTCATGTACA GCACCAAAGG CTTATTTGCG 2100AATAAGAAAA AAAAATGGCA ATTTTACAGT TTCCCAATTG CAATGCATAT TGAATGCAAC 2160AAGCTCTGCC CTTTGAGCTG GCGTATTCAA AGGCAATGCT CCCTCTCCCA AAATTATACG 2220CTGGCTTTCC AAGATGTAGC TGCTTCCATC AATAAACTAT TCTCATTCTG AAAAAAAAAA 2280AAAAAAAAAA AAAAAAAAAA AAAGTCGAC 2309 592 amino acids amino acid linear protein 14Met Ile Gln Ala Leu Leu Val Ile Ile Cys Leu Val Val Phe Pro Tyr1 5 10 15Gln Gly Ser Ser Ile Ile Leu Glu Ser Gly Asn Val Asn Asp Tyr Glu 20 25 30Val Val Tyr Pro Gln Lys Val Pro Ala Leu Leu Lys Gly Gly Val Gln 35 40 45Asn Pro Gln Pro Glu Thr Lys Tyr Glu Asp Thr Met Gln Tyr Glu Phe 50 55 60Gln Val Asn Gly Glu Pro Val Val Leu His Leu Glu Arg Asn Lys Gly65 70 75 80Leu Phe Ser Glu Asp Tyr Thr Glu Thr His Tyr Ala Pro Asp Gly Arg 85 90 95Glu Ile Thr Thr Ser Pro Pro Val Gln Asp His Cys Tyr Tyr His Gly 100 105 110Tyr Ile Gln Asn Glu Ala Asp Ser Ser Ala Ile Ile Ser Ala Cys Asp 115 120 125Gly Leu Lys Gly His Phe Lys His Gln Gly Glu Thr Tyr Phe Ile Glu 130 135 140Pro Leu Lys Leu Phe Asp Ser Glu Ser His Ala Ile Tyr Lys Asp Glu145 150 155 160Asn Val Glu Asn Glu Asp Glu Thr Pro Glu Thr Cys Gly Val Thr Glu 165 170 175Thr Thr Trp Glu Ser Asp Glu Ser Ile Glu Lys Thr Ser Gln Leu Thr 180 185 190Asn Thr Pro Glu Gln Asp Gly Tyr Leu Gln Ala Lys Lys Tyr Ile Glu 195 200 205Phe Tyr Val Val Val Asp Asn Arg Met Tyr Arg Tyr Tyr Lys Arg Asn 210 215 220Glu Pro Ala Ile Lys Arg Arg Val Tyr Glu Met Val Asn Ala Val Asn225 230 235 240Thr Lys Tyr Arg Pro Leu Lys Ile His Ile Thr Leu Ile Gly Leu Glu 245 250 255Ile Trp Ser Asn Asp Asp Lys Phe Glu Val Lys Pro Val Ala Gly Ala 260 265 270Thr Leu Lys Ser Phe Arg Asp Trp Arg Glu Thr Val Leu Leu Pro Arg 275 280 285Lys Arg Asn Asp Asn Ala Gln Leu Leu Thr Gly Ile Asp Phe Asn Gly 290 295 300Thr Val Val Gly Ile Ala Tyr Thr Gly Thr Leu Cys Thr Gln Asn Ser305 310 315 320Val Ala Val Val Gln Asp Tyr Asn Arg Lys Ile Ser Met Val Ala Ser 325 330 335Thr Met Ala His Glu Leu Gly His Asn Leu Gly Leu His His Asp Gly 340 345 350Ala Ser Cys Ile Cys Ser Leu Arg Pro Cys Ile Met Ser Lys Gly Arg 355 360 365Thr Ala Pro Ala Phe Gln Phe Ser Ser Cys Ser Val Arg Glu Tyr Arg 370 375 380Glu Tyr Leu Leu Arg Glu Arg Pro Gln Cys Ile Leu Asn Lys Pro Leu385 390 395 400Ser Thr Asp Thr Val Ser Pro Ala Ile Cys Gly Asn Tyr Phe Val Glu 405 410 415Glu Gly Glu Glu Cys Asp Cys Gly Ser Pro Ala Asp Cys Gln Ser Ala 420 425 430Cys Cys Asp Ala Ala Thr Cys Lys Phe Lys Gly Glu Glu Ala Glu Cys 435 440 445Arg Ala Ala Lys Asp Asp Cys Asp Leu Pro Glu Leu Cys Thr Gly Arg 450 455 460Ser Val Glu Cys Pro Thr Asp Ser Leu Gln Arg Asn Gly His Pro Cys465 470 475 480Gln Asn Asn Lys Gly Tyr Cys Tyr Asn Gly Ala Cys Pro Thr Phe Thr 485 490 495Asn Gln Cys Ile Ala Leu Met Gly Thr Asp Phe Thr Val Ser Pro Asp 500 505 510Gly Cys Phe Asp Leu Asn Val Arg Gly Asn Asp Val Ser His Cys Arg 515 520 525Lys Glu Asn Gly Ala Lys Ile Pro Cys Ala Ala Lys Asp Val Lys Cys 530 535 540Gly Arg Leu Tyr Cys Thr Glu Arg Asp Thr Met Ser Cys Arg Phe Pro545 550 555 560Leu Asp Pro Asp Gly Val Met Ala Glu Pro Gly Thr Lys Cys Gly Asp 565 570 575Gly Met Val Cys Ser Asn Gly Gln Cys Val Asn Val Gln Thr Ala Tyr 580 585 590 1820 base pairs nucleic acid double linear cDNA 15GTCGACGACA TTTCAAGCAT CAAGGGGAGA CATACTTTAT TGAGCCCTTG AAGCTTTTCG 60ACAGTGAATC CCATGCAATC TACAAAGATG AAAATGTAGA AAACGAGGAT GAGACCCCCG 120AAACCTGTGG GGTAACCGAG ACTACTTGGG AGTCAGATGA GTCCATTGAA AAGACCTCTC 180AGTTAACTAA CACTCCTGAA CAAGACGGGT ACTTGCAGGC CAAAAAATAC ATCGAGTTTT 240ACGTGGTTGT GGACAACAGA ATGTACAGGT ATTACAAACG CAATGAACCT GCTATAAAAA 300GAAGAGTATA TGAAATGGTC AACGCTGTAA ATACGAAGTA CAGACCTTTG AAAATTCACA 360TAACACTGAT TGGCCTAGAA ATTTGGTCCA ACGATGATAA GTTTGAAGTG AAGCCAGTAG 420CGGGTGCCAC TTTGAAATCA TTTCGAGATT GGAGAGAAAC AGTTTTGCTG CCACGCAAAA 480GGAATGATAA CGCTCAGTTA CTCACGGGCA TTGACTTCAA TGGAACTGTT GTGGGAATTG 540CTTACACGGG CACCCTCTGC ACTCAGAATT CTGTAGCAGT TGTTCAGGAT TATAACCGAA 600AAATAAGCAT GGTGGCATCT ACAATGGCCC ATGAGTTGGG TCATAATCTG GGCATTCATC 660ATGACGGAGC TTCCTGTATT TGCAGTCTTA AACCATGCAT TATGTCTAAG GGACGGACTG 720CACCTGCCTT TCAGTTCAGC TCTTGTAGTG TCCGGGAGTA TCGGGAGTAT CTTCTTAGAA 780AAAGACCACA ATGCATTCTC AACAAACCCT TGAGCACAGA TATTGTTTCA CCTGCAATTT 840GTGGAAATTA CTTTGTGGAG GAGGGAGAAG AATGTGACTG TGGCTCTCCT GCGGATTGTC 900AAAGTGCCTG CTGCAATGCT GCAACTTGTA AGTTTAAGGG AGAAGAAGCA GAATGCCGGG 960CAGCAAAGGA TGACTGTGAC TTGCCTGAAC TCTGCACTGG CCGATCTGTG GAGTGTCCCA 1020CGGACAGCTT GCAGAGGAAT GGACATCCAT GTCAAAACAA CAAAGGTTAC TGCTACAATG 1080GGGCATGTCC CACCTTCACA AACCAATGTA TTGCTCTCAT GGGGACAGAT TTTACTGTGA 1140GTCCAGATGG ATGTTTTGAC TTGAACGTGA GAGGGAATGA TGTAAGCCAC TGCAGAAAGG 1200AAAATGGTGC AAAGATTCCA TGTGCAGCAA AGGATGTAAA GTGTGGCAGG TTATACTGCA 1260CAGAGAGAAA CACAATGTCA TGCCGATTCC CACTGGACCC AGATGGTGTA ATGGCTGAAC 1320CTGGAACAAA ATGTGGAGAT GGAATGGTGT GCAGCAACGG TCAGTGTGTT AATGTGCAGA 1380CAGCCTACTG ATCAAGCACT GGCTTCTCTC AATTTGATTT TGGAGATCCT CCTTCCAGAA 1440CGCTTCCCTC AAGTCCAAAG AGACCCATCT GTCTTTATCC TACTAGTAAA TCACTCTTAG 1500CTTTCAGATG GTATCTAAAA TTTATAATAT TTCTTCTCCA TAATTTAAAC TGGTAATCTT 1560TTGCTAAAAT CAGACCTTTT CCCTGCCACA AAGCTCCATG GTCATGTACA GTACCAAAGG 1620CTTATTTGCT AACACGAAAA AAAATGGCCA TTTTACCGTT TGCCAATTGC AATTCACATT 1680TAATGCAACA AGCTCTGCCC TTTGAGCTGG CGTATTCAAA GGCAATGCTC CCTCTCCCAA 1740AATTATATGC TGGCTTTCCA AGATGTAGCT GCTTCCATCA ATAAACTATT CTCATTCTGA 1800AAAAAAAAAA AAAAGTCGAC 1820 462 amino acids amino acid linear protein 16Arg Arg His Phe Lys His Gln Gly Glu Thr Tyr Phe Ile Glu Pro Leu1 5 10 15Lys Leu Phe Asp Ser Glu Ser His Ala Ile Tyr Lys Asp Glu Asn Val 20 25 30Glu Asn Glu Asp Glu Thr Pro Glu Thr Cys Gly Val Thr Glu Thr Thr 35 40 45Trp Glu Ser Asp Glu Ser Ile Glu Lys Thr Ser Gln Leu Thr Asn Thr 50 55 60Pro Glu Gln Asp Gly Tyr Leu Gln Ala Lys Lys Tyr Ile Glu Phe Tyr65 70 75 80Val Val Val Asp Asn Arg Met Tyr Arg Tyr Tyr Lys Arg Asn Glu Pro 85 90 95Ala Ile Lys Arg Arg Val Tyr Glu Met Val Asn Ala Val Asn Thr Lys 100 105 110Tyr Arg Pro Leu Lys Ile His Ile Thr Leu Ile Gly Leu Glu Ile Trp 115 120 125Ser Asn Asp Asp Lys Phe Glu Val Lys Pro Val Ala Gly Ala Thr Leu 130 135 140Lys Ser Phe Arg Asp Trp Arg Glu Thr Val Leu Leu Pro Arg Lys Arg145 150 155 160Asn Asp Asn Ala Gln Leu Leu Thr Gly Ile Asp Phe Asn Gly Thr Val 165 170 175Val Gly Ile Ala Tyr Thr Gly Thr Leu Cys Thr Gln Asn Ser Val Ala 180 185 190Val Val Gln Asp Tyr Asn Arg Lys Ile Ser Met Val Ala Ser Thr Met 195 200 205Ala His Glu Leu Gly His Asn Leu Gly Ile His His Asp Gly Ala Ser 210 215 220Cys Ile Cys Ser Leu Lys Pro Cys Ile Met Ser Lys Gly Arg Thr Ala225 230 235 240Pro Ala Phe Gln Phe Ser Ser Cys Ser Val Arg Glu Tyr Arg Glu Tyr 245 250 255Leu Leu Arg Lys Arg Pro Gln Cys Ile Leu Asn Lys Pro Leu Ser Thr 260 265 270Asp Ile Val Ser Pro Ala Ile Cys Gly Asn Tyr Phe Val Glu Glu Gly 275 280 285Glu Glu Cys Asp Cys Gly Ser Pro Ala Asp Cys Gln Ser Ala Cys Cys 290 295 300Asn Ala Ala Thr Cys Lys Phe Lys Gly Glu Glu Ala Glu Cys Arg Ala305 310 315 320Ala Lys Asp Asp Cys Asp Leu Pro Glu Leu Cys Thr Gly Arg Ser Val 325 330 335Glu Cys Pro Thr Asp Ser Leu Gln Arg Asn Gly His Pro Cys Gln Asn 340 345 350Asn Lys Gly Tyr Cys Tyr Asn Gly Ala Cys Pro Thr Phe Thr Asn Gln 355 360 365Cys Ile Ala Leu Met Gly Thr Asp Phe Thr Val Ser Pro Asp Gly Cys 370 375 380Phe Asp Leu Asn Val Arg Gly Asn Asp Val Ser His Cys Arg Lys Glu385 390 395 400Asn Gly Ala Lys Ile Pro Cys Ala Ala Lys Asp Val Lys Cys Gly Arg 405 410 415Leu Tyr Cys Thr Glu Arg Asn Thr Met Ser Cys Arg Phe Pro Leu Asp 420 425 430Pro Asp Gly Val Met Ala Glu Pro Gly Thr Lys Cys Gly Asp Gly Met 435 440 445Val Cys Ser Asn Gly Gln Cys Val Asn Val Gln Thr Ala Tyr 450 455 460 2359 base pairs nucleic acid double linear cDNA 17GTCGACCTCA GGTTGGCTTG GAAGCAGAAA GAGATTCCTA TCCACCACTC CAATCCAGGC 60TCCAAAATGA TCCAAGCTCT CTTGGTAGCT ATATGCTTAG CGGTTTTTCC ATATCAAGGG 120AGCTCTATAA TCCTGGAATC CGGGAATGTT AATGATTATG AAGTAGTGTA TCCACAAAAA 180GTGCCTGCAT TGTCCAAAGG AGGAGTTCAG AATCCTCAGC CAGAGACCAA GTATGAAGAT 240ACAATGCAAT ATGAATTTCA AGTGAATGGA GAGCCAGTAG TCCTTCACCT AGAAAGAAAT 300AAAGGACTTT TTTCAGAAGA TTACACTGAA ACTCATTATG CCTCTGATGG CAGAGAAATT 360ACAACAAGCC CACTCGTTCA GGATCACTGC TATTATCATG GTTACATTCA GAATGAAGCT 420GACTCAAGTG CAGTCATCAG TGCATGCGAT GGCTTGAAAG GACATTTCGA GCTTCAAGGG 480GAGACATACT TTATTGAACC CTTGAAGATT TCCGACAGTG AAGCCCATGC AATCTACAAA 540GATGAAAATG TAGAAAACGA GGATGAGACC CCCGAAACCT GTGGGGTAAC CGAGACTACT 600TGGGAGTCAG ATGAGTCCAT TGAAAAGACC TCTCAGTTAG ACGACGACGA CAAGCGGCCG 660CCAACTAACA CTCCTGAACA AGACAGGTAC TTGCAGGCCA AAAAATACCT CGAGTTTTAC 720GTGGTTGTGG ACAACATAAT GTACAGGCAT TACAAACGCG ATAAACCTGT TATAAAAAGA 780AGAGTATATG AAATGATCAA CACTATGAAT ATGGTGTACA ATCGTTTGAA TTTTCACATA 840GCACTGATTG GCCTAGAAAT TTGGTCCAAC AGAAATGAGA TTAATGTGCA ATCAGACGTG 900CAGGCCACTT TGGACTTATT TGGAGAATGG AGAGAAAAAA AATTGCTGCC ACGCAAAAGG 960AATGATAATG CTCAGTTACT CACGGGTATT GACTTCAAAG GAACTCCTGT AGGACTTGCT 1020TACATAGGTT CCATCTGCAA TCCGAAGAGT TCTGTAGCAG TTGTTCAGGA TTATAGCAGT 1080AGAACAAGCA TGGTGGCAAT TACAATGGCC CATGAGATGG GTCATAATAT GGGCATTCAT 1140CATGACGGAC CTTCCTGTAC TTGTGGTTCT AACAAATGCG TTATGTCTAC AAGACGTACT 1200GAACCTGCCT ATCAGTTCAG CTCTTGTAGT GTCCGGGAAC ATCAGGAGTA TCTTCTTAGA 1260GACAGACCAC AATGCATTCT CAACAAACCC TTGAGCACAG ATATTGTTTC ACCTCCAATT 1320TGTGGAAATA ACTTTGTGGA GGTGGGAGAA GAATGTGACT GTGGCTCTCC TGCGGATTGT 1380CAAAGTGCCT GCTGCGACGC TACAACTTGT AAACTACAAC CTCATGCACA GTGTGACTCC 1440GAAGGGTGTT GTGAGAAATG CAAATTTAAG GGAGCAGGAG CAGAATGCCG GGCAGCAAAG 1500GATGACTGTG ACTTGCCTGA ACTCTGCACT GGCCAATCTG CTGAGTGTCC CACAGACATC 1560TTCCAGAGGA ATGGACTTCC ATGCCAAAAC AACGAAGGTT ACTGCTACAA TGGGAAATGC 1620CCCATCATGA CAAACCAATG TATTGCTCTC CGGGGACCAG GTGTAAAAGT ATCTCGAGAT 1680AGCTGTTTTA CATTGAACCA GAGAACCAGT GGTTGTGGCT TGTGCAGAAT GGAATATGGT 1740AGAAAGATTC CATGTGCAGC AAAGGATGTA AAGTGTGGCA GGTTATTTTG CAAAAAGGGA 1800AACTCGATGA TATGCAACTG CTCAGTTTCA CCACGTGACC CAAGTTATGG AATGGTTGAA 1860CCTGGAACAA AATGTGGAGA TGGAATGGTG TGCAGCAACA GGCAGTGTGT TGATGTGAAG 1920ACAGCCTACT GATCAAGCAC TGGCTTCTCT CAATTTGATT TTGGAGGTCC TCCTTCCAGA 1980ACGCTTCCCT CAAGTCCAAA GAGACCCATC TGTCTTTATC CTACTAGTAA ATCACTCTTA 2040GCTTTCAGAT GGTATCTAAA ATTTAAAATA TTTCTTCTCC ATAATTTAAA CTGGTAATCT 2100TTTGCTAAAA TCAGACCTTT TCCCTGCCAC AAAGCTCCAT GGTCATGTAC AGCACCAAAG 2160GCTTATTTGC TAACAAGAAA AAAAATGGCC ATTTTACTGT TTGCCAATTG CAATTCACAT 2220TTAATGCAAC AAGCTCTGCC CTTTGAGCTG GCGTACTCAA AGGCAATGCT CCCTCTCCCA 2280AAATTATACG CTGGCTTTCC AAGATGTAGC TGCTTCCATC AATAAACTAT TCTCATTCTG 2340AAAAAAAAAA AAAGTCGAC 2359 621 amino acids amino acid linear protein 18Met Ile Gln Ala Leu Leu Val Ala Ile Cys Leu Ala Val Phe Pro Tyr1 5 10 15Gln Gly Ser Ser Ile Ile Leu Glu Ser Gly Asn Val Asn Asp Tyr Glu 20 25 30Val Val Tyr Pro Gln Lys Val Pro Ala Leu Ser Lys Gly Gly Val Gln 35 40 45Asn Pro Gln Pro Glu Thr Lys Tyr Glu Asp Thr Met Gln Tyr Glu Phe 50 55 60Gln Val Asn Gly Glu Pro Val Val Leu His Leu Glu Arg Asn Lys Gly65 70 75 80Leu Phe Ser Glu Asp Tyr Thr Glu Thr His Tyr Ala Ser Asp Gly Arg 85 90 95Glu Ile Thr Thr Ser Pro Leu Val Gln Asp His Cys Tyr Tyr His Gly 100 105 110Tyr Ile Gln Asn Glu Ala Asp Ser Ser Ala Val Ile Ser Ala Cys Asp 115 120 125Gly Leu Lys Gly His Phe Glu Leu Gln Gly Glu Thr Tyr Phe Ile Glu 130 135 140Pro Leu Lys Ile Ser Asp Ser Glu Ala His Ala Ile Tyr Lys Asp Glu145 150 155 160Asn Val Glu Asn Glu Asp Glu Thr Pro Glu Thr Cys Gly Val Thr Glu 165 170 175Thr Thr Trp Glu Ser Asp Glu Ser Ile Glu Lys Thr Ser Gln Leu Asp 180 185 190Asp Asp Asp Lys Arg Pro Pro Thr Asn Thr Pro Glu Gln Asp Arg Tyr 195 200 205Leu Gln Ala Lys Lys Tyr Leu Glu Phe Tyr Val Val Val Asp Asn Ile 210 215 220Met Tyr Arg His Tyr Lys Arg Asp Lys Pro Val Ile Lys Arg Arg Val225 230 235 240Tyr Glu Met Ile Asn Thr Met Asn Met Val Tyr Asn Arg Leu Asn Phe 245 250 255His Ile Ala Leu Ile Gly Leu Glu Ile Trp Ser Asn Arg Asn Glu Ile 260 265 270Asn Val Gln Ser Asp Val Gln Ala Thr Leu Asp Leu Phe Gly Glu Trp 275 280 285Arg Glu Lys Lys Leu Leu Pro Arg Lys Arg Asn Asp Asn Ala Gln Leu 290 295 300Leu Thr Gly Ile Asp Phe Lys Gly Thr Pro Val Gly Leu Ala Tyr Ile305 310 315 320Gly Ser Ile Cys Asn Pro Lys Ser Ser Val Ala Val Val Gln Asp Tyr 325 330 335Ser Ser Arg Thr Ser Met Val Ala Ile Thr Met Ala His Glu Met Gly 340 345 350His Asn Met Gly Ile His His Asp Gly Pro Ser Cys Thr Cys Gly Ser 355 360 365Asn Lys Cys Val Met Ser Thr Arg Arg Thr Glu Pro Ala Tyr Gln Phe 370 375 380Ser Ser Cys Ser Val Arg Glu His Gln Glu Tyr Leu Leu Arg Asp Arg385 390 395 400Pro Gln Cys Ile Leu Asn Lys Pro Leu Ser Thr Asp Ile Val Ser Pro 405 410 415Pro Ile Cys Gly Asn Asn Phe Val Glu Val Gly Glu Glu Cys Asp Cys 420 425 430Gly Ser Pro Ala Asp Cys Gln Ser Ala Cys Cys Asp Ala Thr Thr Cys 435 440 445Lys Leu Gln Pro His Ala Gln Cys Asp Ser Glu Gly Cys Cys Glu Lys 450 455 460Cys Lys Phe Lys Gly Ala Gly Ala Glu Cys Arg Ala Ala Lys Asp Asp465 470 475 480Cys Asp Leu Pro Glu Leu Cys Thr Gly Gln Ser Ala Glu Cys Pro Thr 485 490 495Asp Ile Phe Gln Arg Asn Gly Leu Pro Cys Gln Asn Asn Glu Gly Tyr 500 505 510Cys Tyr Asn Gly Lys Cys Pro Ile Met Thr Asn Gln Cys Ile Ala Leu 515 520 525Arg Gly Pro Gly Val Lys Val Ser Arg Asp Ser Cys Phe Thr Leu Asn 530 535 540Gln Arg Thr Ser Gly Cys Gly Leu Cys Arg Met Glu Tyr Gly Arg Lys545 550 555 560Ile Pro Cys Ala Ala Lys Asp Val Lys Cys Gly Arg Leu Phe Cys Lys 565 570 575Lys Gly Asn Ser Met Ile Cys Asn Cys Ser Val Ser Pro Arg Asp Pro 580 585 590Ser Tyr Gly Met Val Glu Pro Gly Thr Lys Cys Gly Asp Gly Met Val 595 600 605Cys Ser Asn Arg Gln Cys Val Asp Val Lys Thr Ala Tyr 610 615 620 15 base pairs nucleic acid single linear other nucleic acid /desc = “oligonucleotide” 19ACNCCNGARC ARGAY 15 15 base pairs nucleic acid single linear other nucleic acid /desc = “oligonucleotide” 20RTAYTTYCKR TACAT 15 24 base pairs nucleic acid single linear other nucleic acid /desc = “oligonucleotide” 21CAGGACAGGT ACTTGCAGGC CAAA 24 24 base pairs nucleic acid single linear other nucleic acid /desc = “oligonucleotide” 22ATCGAGTTTT ACGTGGTTGT GGAC 24

Claims

1. An isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO:5, or a complement thereof.

2. An isolated polynucleotide consisting of the nucleotide sequence of SEQ ID NO:5, or a complement thereof.

3. An isolated polynucleotide comprising nucleotides 78 to 1940 of SEQ ID NO:5, or a complement thereof.

4. An isolated polynucleotide comprising nucleotides 147 to 1940 of SEQ ID NO:5, or a complement thereof.

5. An isolated polynucleotide comprising nucleotides 651 to 1940 of SEQ ID NO:5, or a complement thereof.

6. An isolated polynucleotide consisting of nucleotides 651 to 1940 of SEQ ID NO:5, or a complement thereof.

7. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence encoded by the cDNA insert of clone NMM-1 deposited with the ATCC as Accession Number 209588.

8. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:6, or a complement thereof.

9. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO:6, or a complement thereof.

10. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising amino acid residues 24 to 621 of SEQ ID NO:6, or a complement thereof.

11. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising amino acid residues 192 to 621 of SEQ ID NO:6, or a complement thereof.

12. The isolated polynucleotide of claim 11, wherein said polynucleotide encodes a polypeptide containing an enterokinase cleavage site.

13. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide consisting of amino acid residues 192 to 621 of SEQ ID NO:6, or a complement thereof.

14. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO:6 having mocarhagin proteolytic activity.

15. The isolated polynucleotide of claim 14, wherein said polynucleotide encodes a polypeptide containing an enterokinase cleavage site.

16. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide consisting of a fragment of the amino acid sequence of SEQ ID NO:6 having mocarhagin proteolytic activity.

17. An isolated polynucleotide comprising a nucleotide sequence encoding a naturally occurring allelic variant of a mocarhagin polypeptide or an active fragment thereof, said mocarhagin polypeptide having mocarhagin proteolytic activity and comprising the amino acid sequence of SEQ ID NO:6.

18. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having mocarhagin proteolytic activity, said polypeptide comprising an amino acid sequence in which at least one amino acid substitution has been introduced compared to the amino acid sequence of SEQ ID NO:6, or an active fragment thereof, wherein said amino acid substitution is at a residue that is not conserved between the mocarhagin polypeptide of SEQ ID NO:6 and a mocarhagin polypeptide selected from the group consisting of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16.

19. The isolated polynucleotide of claim 18, wherein said polynucleotide encodes a polypeptide containing an enterokinase cleavage site.

20. An isolated polynucleotide of any one of claims 1, 2-11, 13, 14, and 16-18, wherein said polynucleotide is operably linked to an expression control sequence.

21. A host cell transformed with a polynucleotide of claim 20.

22. The host cell of claim 21, wherein said cell is a mammalian cell.

23. A process for producing a protein, which comprises:(a) growing a culture of the host cell of claim 21 in a suitable culture medium; and (b) purifying the protein from the culture.

24. An isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO:7, or a complement thereof.

25. An isolated polynucleotide consisting of the nucleotide sequence of SEQ ID NO:7, or a complement thereof.

26. An isolated polynucleotide comprising nucleotides 85 to 1401 of SEQ ID NO:7, or a complement thereof.

27. An isolated polynucleotide comprising nucleotides 154 to 1401 of SEQ ID NO:7, or a complement thereof.

28. An isolated polynucleotide comprising nucleotides 658 to 1401 of SEQ ID NO:7, or a complement thereof.

29. An isolated polynucleotide consisting of nucleotides 658 to 1401 of SEQ ID NO:7, or a complement thereof.

30. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence encoded by the cDNA insert of clone NMM-2 deposited with the ATCC as Accession Number 209589.

31. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:8, or a complement thereof.

32. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO:8, or a complement thereof.

33. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising amino acid residues 24 to 439 of SEQ ID NO:8, or a complement thereof.

34. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising amino acid residues 192 to 439 of SEQ ID NO:8, or a complement thereof.

35. The isolated polynucleotide of claim 34, wherein said polynucleotide encodes a polypeptide containing an enterokinase cleavage site.

36. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide consisting of amino acid residues 192 to 439 of SEQ ID NO:8, or a complement thereof.

37. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO:8 having mocarhagin proteolytic activity.

38. The isolated polynucleotide of claim 37, wherein said polynucleotide encodes a polypeptide containing an enterokinase cleavage site.

39. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide consisting of a fragment of the amino acid sequence of SEQ ID NO:8 having mocarhagin proteolytic activity.

40. An isolated polynucleotide comprising a nucleotide sequence encoding a naturally occurring allelic variant of a mocarhagin polypeptide or an active fragment thereof, said mocarhagin polypeptide having mocarhagin proteolytic activity and comprising the amino acid sequence of SEQ ID NO:8.

41. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having mocarhagin proteolytic activity, said polypeptide comprising an amino acid sequence in which at least one amino acid substitution has been introduced compared to the amino acid sequence of SEQ ID NO:8, or an active fragment thereof, wherein said amino acid substitution is at a residue that is not conserved between the mocarhagin polypeptide of SEQ ID NO:8 and a mocarhagin polypeptide selected from the group consisting of SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16.

42. The isolated polynucleotide of claim 41, wherein said polynucleotide encodes a polypeptide containing an enterokinase cleavage site.

43. An isolated polynucleotide of any one of claims 24, 25-34, 36-37, and 39-41, wherein said polynucleotide is operably linked to an expression control sequence.

44. A host cell transformed with a polynucleotide of claim 43.

45. The host cell of claim 44, wherein said cell is a mammalian cell.

46. A process for producing a protein, which comprises:(a) growing a culture of the host cell of claim 44 in a suitable culture medium; and (b) purifying the protein from the culture.

47. An isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO:9, or a complement thereof.

48. An isolated polynucleotide consisting of the nucleotide sequence of SEQ ID NO:9, or a complement thereof.

49. An isolated polynucleotide comprising nucleotides 67 to 1905 of SEQ ID NO:9, or a complement thereof.

50. An isolated polynucleotide comprising nucleotides 136 to 1905 of SEQ ID NO:9, or a complement thereof.

51. An isolated polynucleotide comprising nucleotides 640 to 1905 of SEQ ID NO:9, or a complement thereof.

52. An isolated polynucleotide consisting of nucleotides 640 to 1905 of SEQ ID NO:9, or a complement thereof.

53. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence encoded by the cDNA insert of clone NMM-9 deposited with the ATCC as Accession Number 209586.

54. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:10, or a complement thereof.

55. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO:10, or a complement thereof.

56. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising amino acid residues 24 to 613 of SEQ ID NO:10, or a complement thereof.

57. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising amino acid residues 192 to 613 of SEQ ID NO:10, or a complement thereof.

58. The isolated polynucleotide of claim 57, wherein said polynucleotide encodes a polypeptide containing an enterokinase cleavage site.

59. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide consisting of amino acid residues 192 to 613 of SEQ ID NO:10, or a complement thereof.

60. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO:10 having mocarhagin proteolytic activity.

61. The isolated polynucleotide of claim 60, wherein said polynucleotide encodes a polypeptide containing an enterokinase cleavage site.

62. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide consisting of a fragment of the amino acid sequence of SEQ ID NO:10 having mocarhagin proteolytic activity.

63. An isolated polynucleotide comprising a nucleotide sequence encoding a naturally occurring allelic variant of a mocarhagin polypeptide or an active fragment thereof, said mocarhagin polypeptide having mocarhagin proteolytic activity and comprising the amino acid sequence of SEQ ID NO:10.

64. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having mocarhagin proteolytic activity, said polypeptide comprising an amino acid sequence in which at least one amino acid substitution has been introduced compared to the amino acid sequence of SEQ ID NO:10, or an active fragment thereof, wherein said amino acid substitution is at a residue that is not conserved between the mocarhagin polypeptide of SEQ ID NO:10 and a mocarhagin polypeptide selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16.

65. The isolated polynucleotide of claim 64, wherein said polynucleotide encodes a polypeptide containing an enterokinase cleavage site.

66. An isolated polynucleotide of any one of claims 47, 48-57, 59-60, and 62-64, wherein said polynucleotide is operably linked to an expression control sequence.

67. A host cell transformed with a polynucleotide of claim 66.

68. The host cell of claim 67, wherein said cell is a mammalian cell.

69. A process for producing a protein, which comprises:(a) growing a culture of the host cell of claim 67 in a suitable culture medium; and (b) purifying the protein from the culture.

70. An isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO:11, or a complement thereof.

71. An isolated polynucleotide consisting of the nucleotide sequence of SEQ ID NO:11, or a complement thereof.

72. An isolated polynucleotide comprising nucleotides 78 to 1640 of SEQ ID NO:11, or a complement thereof.

73. An isolated polynucleotide comprising nucleotides 147 to 1640 of SEQ ID NO:11, or a complement thereof.

74. An isolated polynucleotide comprising nucleotides 651 to 1640 of SEQ ID NO:11, or a complement thereof.

75. An isolated polynucleotide consisting of nucleotides 651 to 1640 of SEQ ID NO:11, or a complement thereof.

76. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence encoded by the cDNA insert of clone NMM-12 deposited with the ATCC as Accession Number 209585.

77. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:12, or a complement thereof.

78. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO:12, or a complement thereof.

79. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising amino acid residues 24 to 521 of SEQ ID NO:12, or a complement thereof.

80. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising amino acid residues 192 to 521 of SEQ ID NO:12, or a complement thereof.

81. The isolated polynucleotide of claim 80, wherein said polynucleotide encodes a polypeptide containing an enterokinase cleavage site.

82. An isolated polynucleotide comprising a nucleotide sequence encoding apolypeptide consisting of amino acid residues 192 to 521 of SEQ ID NO:12, or a complement thereof.

83. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO:12 having mocarhagin proteolytic activity.

84. The isolated polynucleotide of claim 83, wherein said polynucleotide encodes a polypeptide containing an enterokinase cleavage site.

85. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide consisting of a fragment of the amino acid sequence of SEQ ID NO:12 having mocarhagin proteolytic activity.

86. An isolated polynucleotide comprising a nucleotide sequence encoding a naturally occurring allelic variant of a mocarhagin polypeptide or an active fragment thereof, said mocarhagin polypeptide having mocarhagin proteolytic activity and comprising the amino acid sequence of SEQ ID NO:12.

87. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having mocarhagin proteolytic activity, said polypeptide comprising an amino acid sequence in which at least one amino acid substitution has been introduced compared to the amino acid sequence of SEQ ID NO:12, or an active fragment thereof, wherein said amino acid substitution is at a residue that is not conserved between the mocarhagin polypeptide of SEQ ID NO:12 and a mocarhagin polypeptide selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, and SEQ ID NO:16.

88. The isolated polynucleotide of claim 87, wherein said polynucleotide encodes a polypeptide containing an enterokinase cleavage site.

89. An isolated polynucleotide of any one of claims 70, 71-80, 82-83, and 85-87, wherein said polynucleotide is operably linked to an expression control sequence.

90. A host cell transformed with a polynucleotide of claim 89.

91. The host cell of claim 90, wherein said cell is a mammalian cell.

92. A process for producing a protein, which comprises:(a) growing a culture of the host cell of claim 90 in a suitable culture medium; and (b) purifying the protein from the culture.

93. An isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO:13, or a complement thereof.

94. An isolated polynucleotide consisting of the nucleotide sequence of SEQ ID NO:13, or a complement thereof.

95. An isolated polynucleotide comprising nucleotides 83 to 1858 of SEQ ID NO:13, or a complement thereof.

96. An isolated polynucleotide comprising nucleotides 152 to 1858 of SEQ ID NO:13, or a complement thereof.

97. An isolated polynucleotide comprising nucleotides 656 to 1858 of SEQ ID NO:13, or a complement thereof.

98. An isolated polynucleotide consisting of nucleotides 656 to 1858 of SEQ ID NO:13, or a complement thereof.

99. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence encoded by the cDNA insert of clone NMM-13 deposited with the ATCC as Accession Number 209584.

100. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:14, or a complement thereof.

101. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO:14, or a complement thereof.

102. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising amino acid residues 24 to 592 of SEQ ID NO:14, or a complement thereof.

103. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising amino acid residues 192 to 592 of SEQ ID NO:14, or a complement thereof.

104. The isolated polynucleotide of claim 103, wherein said polynucleotide encodes a polypeptide containing an enterokinase cleavage site.

105. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide consisting of amino acid residues 192 to 592 of SEQ ID NO:14, or a complement thereof.

106. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO:14 having mocarhagin proteolytic activity.

107. The isolated polynucleotide of claim 106 wherein said polynucleotide encodes a polypeptide containing an enterokinase cleavage site.

108. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide consisting of a fragment of the amino acid sequence of SEQ ID NO:14 having mocarhagin proteolytic activity.

109. An isolated polynucleotide comprising a nucleotide sequence encoding a naturally occurring allelic variant of a mocarhagin polypeptide or an active fragment thereof, said mocarhagin polypeptide having mocarhagin proteolytic activity and comprising the amino acid sequence of SEQ ID NO:14.

110. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having mocarhagin proteolytic activity, said polypeptide comprising an amino acid sequence in which at least one amino acid substitution has been introduced compared to the amino acid sequence of SEQ ID NO:14, or an active fragment thereof, wherein said amino acid substitution is at a residue that is not conserved between the mocarhagin polypeptide of SEQ ID NO:14 and a mocarhagin polypeptide selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:16.

111. The isolated polynucleotide of claim 110, wherein said polynucleotide encodes a polypeptide containing an enterokinase cleavage site.

112. An isolated polynucleotide of any one of claims 93-103, 105-106, and 108-110, wherein said polynucleotide is operably linked to an expression control sequence.

113. A host cell transformed with a polynucleotide of claim 112.

114. The host cell of claim 113, wherein said cell is a mammalian cell.

115. A process for producing a protein, which comprises:(a) growing a culture of the host cell of claim 113 in a suitable culture medium; and (b) purifying the protein from the culture.

116. An isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO:15, or a complement thereof.

117. An isolated polynucleotide consisting of the nucleotide sequence of SEQ ID NO:15, or a complement thereof.

118. An isolated polynucleotide comprising nucleotides 3 to 1388 of SEQ ID NO:15, or a complement thereof.

119. An isolated polynucleotide comprising nucleotides 186 to 1388 of SEQ ID NO:15, or a complement thereof.

120. An isolated polynucleotide consisting of nucleotides 186 to 1388 of SEQ ID NO:15, or a complement thereof.

121. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence encoded by the cDNA insert of clone NMM-3 deposited with the ATCC as Accession Number 209587.

122. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:16, or a complement thereof.

123. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO:16, or a complement thereof.

124. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising amino acid residues 62 to 462 of SEQ ID NO:16, or a complement thereof.

125. The isolated polynucleotide of claim 124, wherein said polynucleotide encodes a polypeptide containing an enterokinase cleavage site.

126. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide consisting of amino acid residues 62 to 462 of SEQ ID NO:16, or a complement thereof.

127. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO:16 having mocarhagin proteolytic activity.

128. The isolated polynucleotide of claim 127, wherein said polynucleotide encodes a polypeptide containing an enterokinase cleavage site.

129. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide consisting of a fragment of the amino acid sequence of SEQ ID NO:16 having mocarhagin proteolytic activity.

130. An isolated polynucleotide comprising a nucleotide sequence encoding a naturally occurring allelic variant of a mocarhagin polypeptide or an active fragment thereof, said mocarhagin polypeptide having mocarhagin proteolytic activity and comprising the amino acid sequence of SEQ ID NO:16.

131. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having mocarhagin proteolytic activity, said polypeptide comprising an amino acid sequence in which at least one amino acid substitution has been introduced compared to the amino acid sequence of SEQ ID NO:16, or an active fragment thereof, wherein said amino acid substitution is at a residue that is not conserved between the mocarhagin polypeptide of SEQ ID NO:16 and a mocarhagin polypeptide selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:1O, SEQ ID NO:12, and SEQ ID NO:14.

132. The isolated polynucleotide of claim 131, wherein said polynucleotide encodes a polypeptide containing an enterokinase cleavage site.

133. An isolated polynucleotide of any one of claims 116-124, 126-127, and 129-131, wherein said polynucleotide is operably linked to an expression control sequence.

134. A host cell transformed with a polynucleotide of claim 133.

135. The host cell of claim 134, wherein said cell is a mammalian cell.

136. A process for producing a protein, which comprises:(a) growing a culture of the host cell of claim 134 in a suitable culture medium; and (b) purifying the protein from the culture.

137. An isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO:17, or a complement thereof.

138. An isolated polynucleotide consisting of the nucleotide sequence of SEQ ID NO:17, or a complement thereof.

139. An isolated polynucleotide comprising nucleotides 67 to 1929 of SEQ ID NO:17, or a complement thereof.

140. An isolated polynucleotide comprising nucleotides 655 to 1929 of SEQ ID NO:17, or a complement thereof.

141. An isolated polynucleotide consisting of nucleotides 655 to 1929 of SEQ ID NO:17, or a complement thereof.

142. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence encoded by the cDNA insert of clone NMM-9ek deposited with the ATCC as Accession Number 209583.

143. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:18, or a complement thereof.

144. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO:18, or a complement thereof.

145. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising amino acid residues 197 to 621 of SEQ ID NO:18, or a complement thereof.

146. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide consisting of amino acid residues 197 to 621 of SEQ ID NO:18, or a complement thereof.

147. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO:18 having mocarhagin proteolytic activity.

148. The isolated polynucleotide of claim 147, wherein said polynucleotide encodes a polypeptide containing an enterokinase cleavage site.

149. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide consisting of a fragment of the amino acid sequence of SEQ ID NO:18 having mocarhagin proteolytic activity.

150. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having mocarhagin proteolytic activity, said polypeptide comprising an amino acid sequence in which at least one amino acid substitution has been introduced compared to the amino acid sequence of SEQ ID NO:18, or an active fragment thereof, wherein said amino acid substitution is at a residue that is not conserved between the mocarhagin polypeptide of SEQ ID NO:18 and a mocarhagin polypeptide selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:I0, SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16.

151. The isolated polynucleotide of claim 150, wherein said polynucleotide encodes a polypeptide containing an enterokinase cleavage site.

152. An isolated polynucleotide comprising a nucleotide sequence encoding a naturally occurring allelic variant of a mocarhagin polypeptide or an active fragment thereof, said mocarhagin polypeptide having mocarhagin proteolytic activity and comprising the amino acid sequence of SEQ ID NO:18.

153. An isolated polynucleotide of any one of claims 137-147, 149-150, and 152, wherein said polynucleotide is operably linked to an expression control sequence.

154. A host cell transformed with a polynucleotide of claim 153.

155. The host cell of claim 154, wherein said cell is a mammalian cell.

156. A process for producing a protein, which comprises:(a) growing a culture of the host cell of claim 154 in a suitable culture medium; and (b) purifying the protein from the culture.