IP Library › Granted Patent US 6,881,404

Granted Patent B2

US 6,881,404 · App. 09/996,620 · Granted Apr 19, 2005

Highly purified mocarhagin, a cobra venom protease, polynucleotides encoding same and related proteases, and therapeutic uses thereof

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Quick Facts

Patent No.: US 6,881,404
App. No.: 09/996,620
Granted: Apr 19, 2005
Kind: B2

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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.

Claims (115)

1. A composition comprising a mature mocarhagin protein at least 95% free of other cobra proteins and comprising an N terminal amino acid sequence chosen from:

a) amino acids 192 to 221 of SEQ ID NO: 6;

b) amino acids 192 to 221 of SEQ ID NO: 8;

c) amino acids 192 to 221 of SEQ ID NO: 10;

d) amino acids 192 to 221 of SEQ ID NO: 12;

e) amino acids 192 to 221 of SEQ ID NO: 14.

2. The composition of claim 1 , wherein said mocarhagin protein exhibits an IC50 of less than about 100 μg/mL in a neutrophil/HL60 binding inhibition assay.

3. The composition of claim 1 , wherein said 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;

(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.

4. The composition of claim 1 , wherein said mocarhagin protein is capable of cleaving a material selected from the group consisting of anionic polypeptides containing sulfated tyrosine residues, PSGL-1 and GP Ibα.

5. A composition comprising a therapeutically effective amount of a composition of claim 1 , and a pharmaceutically acceptable carrier.

6. A composition comprising a mature mocarhagin protein isolated from cobra venom by:

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;

(d) eluting said mocarhagin from said Mono S column; and wherein and the mature mocarhagin protein comprises an N terminal amino acid sequence chosen from:

a) amino acids 192 to 221 of SEQ ID NO: 6;

b) amino acids 192 to 221 of SEQ ID NO: 8;

c) amino acids 192 to 221 of SEQ ID NO: 10;

d) amino acids 192 to 221 of SEQ ID NO: 12;

e) amino acids 192 to 221 of SEQ ID NO: 14.

7. The composition of claim 6 further comprising a pharmaceutically acceptable carrier.

8. The composition of claim 2 , wherein said mocarhagin protein exhibits an IC50 of less than about 1 μg/mL in a neutrophil/HL60 binding inhibition assay.

9. The composition of claim 1 , wherein said mocarhagin protein is homogeneous.

10. The composition of claim 3 , wherein said protein comprises the amino acid sequence of SEQ ID NO:6 from amino acid 192 to amino acid 621.

11. 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;

(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.

12. The composition of claim 11 , wherein said protein comprises the amino acid sequence of SEQ ID NO:6.

13. A protein produced according to a process comprising:

(a) in a suitable culture medium, growing a culture of a host cell transformed with an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 operably linked to an expression control sequence; and

(b) purifying the protein from the culture.

14. The protein of claim 13 comprising a mature protein.

15. A pharmaceutical composition comprising a protein of claim 11 and a pharmaceutically acceptable carrier.

16. 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;

(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.

17. The composition of claim 16 , wherein said protein comprises the amino acid sequence of SEQ ID NO: 8.

18. A protein produced according to a process comprising:

(a) in a suitable culture medium, growing a culture of a host cell transformed with an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO: 7 operably linked to an expression control sequence; and

(b) purifying the protein from the culture.

19. The protein of claim 18 comprising a mature protein.

20. A pharmaceutical composition comprising a protein of claim 16 and a pharmaceutically acceptable carrier.

21. 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;

(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.

22. The composition of claim 21 , wherein said protein comprises the amino acid sequence of SEQ ID NO: 10.

23. A protein produced according to a process comprising:

(a) in a suitable culture medium, growing a culture of a host cell transformed with an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO: 9 operably linked to an expression control sequence; and

(b) purifying the protein from the culture.

24. The protein of claim 23 comprising a mature protein.

25. A pharmaceutical composition comprising a protein of claim 21 and a pharmaceutically acceptable carrier.

26. 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;

(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.

27. The composition of claim 26 , wherein said protein comprises the amino acid sequence of SEQ ID NO: 12.

28. A protein produced according to a process comprising:

(a) in a suitable culture medium, growing a culture of a host cell transformed with an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO: 11 operably linked to an expression control sequence; and

(b) purifying the protein from the culture.

29. The protein of claim 28 comprising a mature protein.

30. A pharmaceutical composition comprising a protein of claim 26 and a pharmaceutically acceptable carrier.

31. 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;

(d) fragments of the amino acid sequence of SEQ ID NO: 14 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.

32. The composition of claim 31 wherein said protein comprises the amino acid sequence of SEQ ID NO: 14.

33. A protein produced according to a process comprising:

(a) in a suitable culture medium, growing a culture of a host cell transformed with an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO: 13 operably linked to an expression control sequence; and

(b) purifying the protein from the culture.

34. The protein of claim 33 comprising a mature protein.

35. A pharmaceutical composition comprising a protein of claim 31 and a pharmaceutically acceptable carrier.

36. 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;

(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.

37. The composition of claim 36 , wherein said protein comprises the amino acid sequence of SEQ ID NO: 16.

38. A protein produced according to a process comprising:

(a) in a suitable culture medium, growing a culture of a host cell transformed with an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO: 15 operably linked to an expression control sequence; and

(b) purifying the protein from the culture.

39. The protein of claim 38 comprising a mature protein.

40. A pharmaceutical composition comprising a protein of claim 36 and a pharmaceutically acceptable carrier.

41. 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;

(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.

42. The composition of claim 41 , wherein said protein comprises the amino acid sequence of SEQ ID NO: 18.

43. A protein produced according to a process comprising:

(a) in a suitable culture medium, growing a culture of a host cell transformed with an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO:17 operably linked to an expression control sequence; and

(b) purifying the protein from the culture.

44. The protein of claim 43 comprising a mature protein.

45. A pharmaceutical composition comprising a protein of claim 41 and a pharmaceutically acceptable carrier.

Assignments (1)

CHANGE OF NAME Recorded Mar 29, 2002

From: GENETICS INSTITUTE, INC.

To: GENETICS INSTITUTE, LLC

Reel/Frame 012772/0631 →