Method for making insulin precursors and insulin precursor analogues having improved fermentation yield in yeast

• US 6,777,207 B2
• Filed: 06/28/2001
• Issued: 08/17/2004
• Est. Priority Date: 12/29/1999
• Status: Expired due to Term

• Current Assignee: Novo Nordisk A/S
• Original Assignee: Novo Nordisk A/S
• Inventors: Ludvigsen, Svend, Kjeldsen, Thomas Børglum
• Primary Examiner(s): MOSHER, MARY
• Application Number:
  US09/894,711
• Publication Number:
  US 20020137144A1
• Time in Patent Office: 1,146 Days
• Field of Search: 530/303, 530/305, 530/350, 435/69.4, 435/320.1, 435/252.3, 435/254.2, 435/254.21, 435/325, 435/69.1, 435/69.7, 536/23.51, 536/23.4, 536/23.5
• US Class Current: 435/69.4
• CPC Class Codes: C07K 14/62 Insulins

Abstract

Novel insulin precursors and insulin precursor analogs comprising a connecting peptide (mini C-peptide) of preferably up to 15 amino acid residues and comprising at least one Gly are provided. The precursors can be converted into human insulin or a human insulin analog. The precursors will typically have a distance between B27 (atom CG2) and A1 (atom CA) of less than 5 Å.

Claims

1. An insulin precursor or insulin analog precursor comprising a sequence of formula I:

2. An insulin precursor or an insulin analog precursor according to claim 1, wherein X₁ is 1-15 amino acid residues in length.

3. An insulin precursor or an insulin analog precursor according to claim 1, wherein X₁ is 1-10 amino acid residues in length.

4. An insulin precursor or an insulin analog precursor according to claim 1, wherein X₁ is 1-8 amino acid residues in length.

5. An insulin precursor or an insulin analog precursor according to claim 1, wherein X₁ is 1-5 amino acid residues in length.

6. An insulin precursor or an insulin analog precursor according to claim 1, wherein X₁ is 1-3 amino acid residues in length.

7. An insulin precursor or an insulin analog precursor according to claim 1, wherein X₁ contains 1-5 Gly.

8. An insulin precursor or an insulin analog precursor according to claim 1, wherein X₁ contains 1-3 Gly.

9. An insulin precursor or an insulin analog precursor according to claim 1, wherein X₃ is Asp and X₂ is Lys.

10. An insulin precursor or an insulin analog precursor according to claim 1, wherein X₁—

Y is selected from the group of;

(a) Glu-Glu-Gly-Lys(SEQ ID NO;

1) (b) Glu-Gly-Lys, (c) Ser-Gly-Lys, (d) Asn-Gly-Lys, (e) Thr-Gly-Lys, (f) Asp-Gly-Lys, (g) Met-Gly-Lys, (h) Ala-Gly-Lys, (i) His-Gly-Lys and (j) Gly-Lys.

11. An insulin precursor or insulin analog precursor according to claim 1, wherein the B27 (atom CG2) has a proximity to the A1 (atom CA) of less than 5 Å

.

12. A polynucleotide sequence encoding an insulin precursor or an insulin analog precursor according to claim 1.

13. An expression vector comprising a polynucleotide sequence according to claim 12.

14. A host cell transformed with the vector of claim 13.

15. A process for making an insulin precursor or an insulin analog precursor, said method comprising (i) culturing a host comprising a polynucleotide sequence according to claim 12 under suitble culture conditions for expression of said precursor;

and (ii) isolating the expressed precursor.

16. A process according to claim 15, wherein the host cell is a yeast host cell.

17. A process according to claim 15, further comprising (iii) converting the precursor into insulin or an insulin analog by in vitro chemical or enzymatic conversion.

18. An insulin precursor or insulin analog precursor comprising a connecting peptide (C-peptide) being cleavable from the A and B chains said connecting peptide comprising at least one Gly and a cleavage site enabling cleavage of the peptide bond between the A-chain and the connecting peptide, wherein (i) one Gly is immediately N-terminal to said cleavage site and (ii) said connecting peptide does not contain two adjacent basic amino acids.

19. An insulin precursor or insulin analog precursor according to claim 18, wherein the connecting pepride is 1-15 amino acid residues in length.

20. An insulin precursor or insulin analog precursor according to claim 18, wherein the connecting peptide is 1-10 amino acid residues in length.

21. An insulin precursor or insulin analog precursor according to claim 18, wherein the connecting peptide is 1-9 amino acid residues in length.

22. An insulin precursor or insulin analog precursor according to claim 18, wherein the connecting peptide is 1-5 amino acid residues in length.

23. An insulin precursor or insulin analog precursor according to claim 18, wherein the connecting peptide is 1-3 amino acid residues in length.

24. An insulin precursor or insulin analog precursor according to claim 18, wherein the B27 (atom CG2) has a proximity to the A1 (atom CA) of less than 5 Å

.

25. An insulin precursor or insulin analog precursor according to claim 18, wherein the connecting peptide contains 1-5 Gly.

26. An insulin precursor or insulin analog precursor according to claim 18, wherein the connecting peptide contains 1-3 Gly.

27. A polynucleotide sequence encoding an insulin precursor or an insulin analog precursor according to claim 18.

28. An expression vector comprising a polynucleotide sequence according to claim 27.

29. A host cell transformed with the vector of claim 28.

30. A process for making an insulin precursor or an insulin analog precursor, said method comprising (i) culturing a host cell comprising a polynucleotide sequence according to claim 27 under suitable culture conditions for expression of said precursor;

and (ii) isolating the expressed precursor.

31. A process according to claim 30, wherein the host cell is a yeast host cell.

32. A process according to claim 30, further comprising (iii) converting the precursor into insulin or an insulin analog by in vitro chemical or enzymatic conversion.