Acid-cleavable linkers exhibiting altered rates of acid hydrolysis

• US 8,609,621 B2
• Filed: 09/07/2011
• Issued: 12/17/2013
• Est. Priority Date: 11/15/2010
• Status: Expired due to Fees

• Current Assignee: Dupont US Holding, LLC (International Flavors & Fragrances Incorporated)
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Bedzyk, Laura A., Fahnestock, Stephen R., Gruber, Tanja Maria, Okeefe, Daniel P., Rouviere, Pierre E.
• Primary Examiner(s): HEARD, THOMAS SWEENEY
• Application Number:
  US13/226,610
• Publication Number:
  US 20120122153A1
• Time in Patent Office: 832 Days
• Field of Search: None
• US Class Current: 514/21.7
• CPC Class Codes: C07K 1/12 by hydrolysis , i.e. solvolysis in general
• CPC Class Codes: C07K 14/435 from animals; from humans
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/01 containing a localisation/targetting motif
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: C07K 5/1021 with the first amino acid being acidic
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12P 21/06 produced by the hydrolysis of a peptide bond, e.g. hydrolysate products preparing foodstuffs by protein hydrolysis A23J3/00

Abstract

An acid-cleavable peptide linker comprising aspartic acid and proline residues is disclosed. The acid-cleavable peptide linker provides an altered sensitivity to acid-hydrolytic release of peptides of interest from fusion peptides of the formula PEP1-L-PEP2. The inventive linker, L, is described in various embodiments, each of which provides substantially more rapid acid-release of peptides of interest than does a single aspartic acid-proline pair. In an additional aspect, a method of increasing the stability of an acid cleavable linkage to acid hydrolysis is also provided.

Claims

1. A method of preparing at least one peptide of interest (“

POI”

) from a fusion peptide comprising at least one POI, comprising;

a) providing a recombinant cell synthesizing a fusion peptide having the structure

PEP1-L-PEP2

wherein,

i) PEP1 and PEP2 are independently functional peptides wherein at least one is a peptide of interest (“

POI”

); and

ii) L is an acid-cleavable linker comprising a peptide

wherein D is aspartic acid and P is proline;

b) contacting the fusion peptide with a solution of sufficiently acidic pH so that linker L is cleaved, and

c) isolating the at least one POI.

2. The method of claim 1 wherein the recombinant cell is a recombinant microbial cell.

3. The method of claim 2 wherein the recombinant microbial cell is a recombinant yeast cell.

4. The method of claim 2 wherein the recombinant microbial cell is a recombinant bacterial cell.

5. The method of claim 1 wherein the acid-cleavable linker is cleaved by incubating the fusion peptides at a pH in the range from about pH 1 to about pH 4.

6. The method of claim 1 wherein the acid-cleavable linker is cleaved by incubating the fusion peptides at a pH in the range from about pH 2 to about pH 4.

7. The method of claim 1 wherein the acid-cleavable linker is cleaved by incubating the fusion peptides at a pH in the range from about pH 3 to about pH 4.

8. The method of claim 1 wherein the acid-cleavable linker is cleaved by incubating the fusion peptides at a pH of about 4.

9. The method of claim 1 wherein the acid-cleavable linker is cleaved by incubating the fusion peptides at a temperature of about 40°

C. to about 90°

C.

10. The method of claim 1 wherein the acid-cleavable linker is cleaved by incubating the fusion peptides at a temperature of about 50°

C. to about 80°

C.

11. The method of claim 1 wherein the acid-cleavable linker is cleaved by incubating the fusion peptides at a temperature of about 60°

C. to about 70°

C.

12. The method of claim 1 wherein the acid-cleavable linker is cleaved by incubating the fusion peptides at a temperature of about 60°

C.

13. The method of claim 1 wherein the acid-cleavable linker is cleaved by incubating the fusion peptides at a pH of about pH 2 to about pH 4 using a temperature of about 50°

C. to about 80°

C.

14. The method of claim 1, wherein PEP1 and PEP2 are both POIs.

15. The method of claim 14, wherein the fusion peptide is soluble in the recombinant cell.

16. The method of claim 14, wherein the fusion peptide is insoluble in the recombinant cell.

17. The method of claim 16, wherein cleaving the fusion peptide under acidic conditions renders the at least one POI soluble.

18. The method of claim 1, wherein either PEP1 or PEP2 of the fusion peptide comprises an inclusion body tag, thereby comprising a non-POI portion of the fusion peptide.

19. The method of claim 18, wherein the non-POI portion remains insoluble after cleaving the fusion peptide.

20. A fusion peptide comprising two peptides separated by an acid-cleavable linker according to the following general formula

PEP1-L-PEP

wherein,

a) PEP1 and PEP2 are independently functional peptides wherein at least one is a peptide of interest (“

POI”

); and

b) L is an acid-cleavable linker comprising a peptide

wherein D is aspartic acid and P is proline.

21. The fusion peptide of claim 20 wherein PEP1 and PEP2 are nonidentical.

22. The fusion peptide of claim 21, wherein the fusion peptide is soluble in a recombinant cell.

23. The fusion peptide of claim 22 wherein the recombinant cell is a recombinant microbial cell.

24. The fusion peptide of claim 23 wherein the recombinant microbial cell is a recombinant bacterial cell.

25. The fusion peptide of claim 23 wherein the recombinant microbial cell is a recombinant yeast cell.

26. The fusion peptide of claim 21, wherein the fusion peptide is insoluble in a recombinant cell.

27. The fusion peptide of claim 26 wherein the recombinant cell is a recombinant microbial cell.

28. The fusion peptide of claim 27 wherein the recombinant microbial cell is a recombinant yeast cell.

29. The fusion peptide of claim 28 wherein the recombinant microbial cell is a recombinant bacterial cell.

30. The fusion peptide of claim 21 wherein either of PEP1 or PEP2 comprises an inclusion body tag (“

IBT”

).

31. The fusion peptide of claim 30 wherein the fusion peptide is present in inclusion bodies.

32. The fusion peptide of claim 21 wherein the acid-cleavable linker is cleaved by incubation at a pH in the range from about pH 1 to about pH 4.

33. The fusion peptide of claim 21 wherein the acid-cleavable linker is cleaved by incubating at a temperature of about 40°

C. to about 90°

C.

34. The fusion peptide of claim 33 wherein the acid-cleavable linker is cleaved by incubating at a temperature of about 50°

C. to about 80°

C.

35. The fusion peptide of claim 34 wherein the acid-cleavable linker is cleaved by incubating at a temperature of about 60°

C. to about 70°

C.

36. The fusion peptide of claim 21 wherein the acid-cleavable linker is cleaved by incubating at a pH of about pH 2 to about pH 4 and at a temperature of about 50°

C. to about 80°

C.

37. A recombinant cell expressing a fusion protein having the structure

PEP1-L-PEP2

wherein,

i) PEP1 and PEP2 are independently functional peptides, one of which is a POI;

and

ii) L is an acid-cleavable linker comprising a peptide

wherein D is aspartic acid and P is proline; and

wherein the expressed fusion peptide is present in the recombinant cell.

38. The recombinant cell of claim 37 wherein the recombinant cell is a recombinant microbial cell.

39. The recombinant cell of claim 38 wherein the recombinant cell is a recombinant bacterial cell.

40. The recombinant cell of claim 38 wherein the recombinant cell is a recombinant microbial cell is a recombinant yeast cell.

41. An acid-cleavable peptide linker comprising a peptide

wherein D is aspartic acid and P is proline.