Claims
- 1. A method of producing an insulin C-peptide, which comprises expressing in a host cell a multimeric polypeptide comprising multiple copies of a said insulin C-peptide, and cleaving said expressed polypeptide to release single copies of the insulin C-peptide.
- 2. A nucleic acid molecule comprising multiple copies of a nucleotide sequence encoding an insulin C-peptide, wherein said nucleic acid molecule encodes a multimeric polypeptide capable of being cleaved to yield single copies of said insulin C-peptide.
- 3. A method for the production of a nucleic acid molecule which encodes a multimeric polypeptide comprising multiple copies of an insulin C-peptide, wherein the expressed multimeric polypeptide is capable of being subsequently cleaved to yield single copies of the insulin C-peptide, said process comprising generating a nucleic acid molecule comprising multiple copies of a nucleotide sequence encoding an insulin C-peptide, linked in matching reading frame.
- 4. A multimeric polypeptide comprising multiple copies of an insulin C-peptide, wherein said multimeric polypeptide can be cleaved to release single copies of said insulin C-peptide.
- 5. A method of producing a multimeric polypeptide which contains multiple copies of an insulin C-peptide and can be cleaved to release single copies of said insulin C-peptide, said method comprising culturing a host cell containing a nucleic acid molecule encoding said multimeric polypeptide under conditions whereby said multimeric polypeptide is expressed, and recovering the expressed multimeric polypeptide.
- 6. A method of producing an insulin C-peptide, said method comprising cleaving a multimeric polypeptide as defined in claim 4.
- 7. A method according to claim 1, wherein said multiple copies of said insulin C-peptide are arranged in tandem.
- 8. A method according to claim 1, wherein said multimeric polypeptide comprises 2 to 30 copies of said insulin C-peptide.
- 9. A method according to claim 8, wherein said multimeric polypeptide comprises 3 to 7 copies of said insulin C-peptide.
- 10. A method according to claim 1, wherein said multimeric polypeptide further comprises a fusion partner.
- 11. A method according to claim 10, wherein said fusion partner is one of a pair of affinity binding partners or ligands.
- 12. A method according to claim 11, wherein said fusion partner is the 25 kDa serum albumin binding region (BB) derived from streptococcal protein G.
- 13. A method according to claim 1, wherein the insulin C-peptide monomers in said multimeric polypeptide are flanked by linker regions comprising a cleavage site.
- 14. A method according to claim 13, wherein said cleavage site is cleavable by a proteolytic enzyme.
- 15. A method according to claim 14, wherein said cleavage site comprises arginine residues for cleavage by trypsin and carboxypeptidase B.
- 16. An expression vector comprising a nucleic acid molecule as defined in claim 2.
- 17. The expression vector according to claim 16, said expression vector being a plasmid.
- 18. The expression vector according to claim 17, wherein said expression vector is based on plasmid pTrpBB (SEQ ID NO: 14).
- 19. A host cell containing a nucleic acid molecule as defined in claim 2.
- 20. An insulin C-peptide produced by the method of claim 1.
- 21. An insulin C-peptide produced by the method of claim 6.
- 22. The nucleic acid molecule according to claim 2, wherein said multiple copies of said insulin C-peptide or said insulin C-peptide encoding-nucleotide sequence are arranged in tandem.
- 23. The nucleic acid molecule according to claim 2, wherein said multimeric polypeptide comprises 2 to 30 copies of said insulin C-peptide.
- 24. The nucleic acid molecule according to claim 23, wherein said multimeric polypeptide comprises 3 to 7 copies of said insulin C-peptide.
- 25. The nucleic acid molecule according to claim 2, wherein said multimeric polypeptide further comprises a fusion partner.
- 26. The nucleic acid molecule according to claim 25, wherein said fusion partner is an affinity binding partner or a ligand.
- 27. The nucleic acid molecule according to claim 26, wherein said fusion partner is a 25 kDa serum albumin binding region (BB) derived from streptococcal protein G.
- 28. The nucleic acid molecule according to claim 2, wherein each insulin C-peptide in said multimeric polypeptide is flanked by linker regions comprising a cleavage site.
- 29. A nucleic acid molecule according to claim 28, wherein said cleavage site is cleavable by a proteolytic enzyme.
- 30. A nucleic acid molecule according to claim 29, wherein said cleavage site comprises arginine residues for cleavage by trypsin and carboxypeptidase B.
- 31. A nucleic acid molecule according to claim 2, wherein said nucleic acid molecule further comprises one or more regulatory or expression control sequences.
- 32. The nucleic acid molecule according to claim 2, wherein said multiple copies of said nucleotide sequence encoding said insulin C-peptide are in matching reading frame.
- 33. The multimeric polypeptide according to claim 4, wherein said multiple copies of said insulin C-peptide are arranged in tandem.
- 34. The multimeric polypeptide according to claim 4, wherein said multimeric polypeptide comprises 2 to 30 copies of said insulin C-peptide.
- 35. The multimeric polypeptide according to claim 34, wherein said multimeric polypeptide comprises 3 to 7 copies of said insulin C-peptide.
- 36. The multimeric polypeptide according to claim 4, wherein said multimeric polypeptide further comprises a fusion partner.
- 37. The multimeric polypeptide according to claim 36, wherein said fusion partner is an affinity binding partner or a ligand.
- 38. The multimeric polypeptide according to claim 37, wherein said fusion partner is a 25 kDa serum albumin binding region (BB) derived from streptococcal protein G.
- 39. The multimeric polypeptide according to claim 4, wherein each insulin C-peptide in said multimeric polypeptide is flanked by linker regions comprising a cleavage site.
- 40. The multimeric polypeptide according to claim 39, wherein said cleavage site is cleavable by a proteolytic enzyme.
- 41. The multimeric polypeptide according to claim 40, wherein said cleavage site comprises arginine residues for cleavage by trypsin and carboxypeptidase B.
- 42. The method according to claim 5, wherein said multiple copies of said insulin C-peptide are arranged in tandem.
- 43. The method according to claim 5, wherein said multimeric polypeptide comprises 2 to 30 copies of said insulin C-peptide.
- 44. The method according to claim 43, wherein said multimeric polypeptide comprises 3 to 7 copies of said insulin C-peptide.
- 45. The method according to claim 5, wherein said multimeric polypeptide further comprises a fusion partner.
- 46. The method according to claim 45, wherein said fusion partner is an affinity binding partner or a ligand.
- 47. The method according to claim 46, wherein said fusion partner is a 25 kDa serum albumin binding region (BB) derived from streptococcal protein G.
- 48. The method according to claim 5, wherein each insulin C-peptide in said multimeric polypeptide is flanked by linker regions comprising a cleavage site.
- 49. The method according to claim 48, wherein said cleavage site is cleavable by a proteolytic enzyme.
- 50. The method according to claim 49, wherein said cleavage site comprises arginine residues for cleavage by trypsin and carboxypeptidase B.
Priority Claims (1)
Number |
Date |
Country |
Kind |
9716790.2 |
Aug 1997 |
GB |
|
Parent Case Info
[0001] This application is a continuation-in-part of U.S. application Ser. No. 09/485,286, filed Feb. 7, 2000, which is the National Stage of International Application No. PCT/GB98/02382, filed Aug. 8, 1998, which claims foreign priority from British patent Application No. 9716790.2, filed Aug. 7, 1997, the contents of which are incorporated here by reference.
Continuation in Parts (1)
|
Number |
Date |
Country |
Parent |
09485286 |
Feb 2000 |
US |
Child |
10430752 |
May 2003 |
US |