Method of expressing proteins with disulfide bridges

• US 8,685,668 B2
• Filed: 11/20/2012
• Issued: 04/01/2014
• Est. Priority Date: 02/11/2005
• Status: Active Grant

• Current Assignee: University of Southern California
• Original Assignee: University of Southern California
• Inventors: Minea, Radu O., Markland, Francis S. Jr.
• Primary Examiner(s): Noakes, Suzanne M
• Assistant Examiner(s): Lee, Jae W
• Application Number:
  US13/682,470
• Publication Number:
  US 20130345399A1
• Time in Patent Office: 497 Days
• Field of Search: None
• US Class Current: 435/69.1
• CPC Class Codes: A61K 38/1703 from vertebrates
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/04 specific for metastasis
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/02 Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: C07K 14/245 Escherichia (G)
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/21 containing a His-tag
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: C07K 2319/90 containing a motif for post-translational modification
• CPC Class Codes: C12N 9/6418 from snakes
• CPC Class Codes: C12N 9/90 Isomerases (5.)
• CPC Class Codes: C12P 21/00 Preparation of peptides or proteins single cell protein C12N1/00
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione

Abstract

This invention relates to methods of expressing eukaryotic proteins in prokaryotic hosts, particularly eukaryotic proteins that require formation of disulfide bridges for biological activity. Various approaches are used including fusion to thioredoxin, cytoplasmic expression of disulfide isomerases, deficiencies in thioredoxin and/or glutathione reductases, deficiencies in proteases, and the like. The method is applicable to express monomeric and dimeric forms of the eukaryotic protein with biological activity such as monomeric and dimeric forms of a disintegrin or a disintegrin domain. Included are the vectors, host cells expressing the proteins, the expressed proteins and methods of using the proteins.

Claims

1. A method of expressing in prokaryotic host cells a eukaryotic protein that requires formation of multiple interchain or intrachain disulfide bridges for its biological activity, said method comprising:

a) transforming a prokaryotic host cell with an expression vector encoding a fusion protein which comprises an N-terminal segment encoding thioredoxin and a C-terminal segment encoding a eukaryotic protein that requires formation of at least two intrachain or interchain disulfide bridges for biological activity; and

b) expressing said eukaryotic protein fused to said thioredoxin in biologically active form,

wherein the host cell has been modified to express a disulfide isomerase DsbC in the cytoplasm of the cell, and

wherein the DsbC has the amino acid sequence as set forth in SEQ ID NO;

3 which is modified so that its active site sequence between positions 98-101 has been changed to CGFC (SEQ ID NO;

     45) or CTFC (SEQ ID NO;

     46).

2. The method of claim 1 wherein the host cell has been modified to express a thiol disulfide interchange protein (DsbD) alpha domain fragment in the cytoplasm.

3. The method of claim 2 wherein the DsbD alpha domain fragment consist of the amino acid sequence as set forth in SEQ ID NO:

4.

4. The method of claim 1 wherein the thioredoxin portion of the fusion protein is from the same type organism as the prokaryotic host.

5. The method of claim 1 wherein the thioredoxin portion of the fusion protein is selected from the group of:

the amino acid sequence as set forth in SEQ ID NO;

1 which contains CPYC SEQ ID NO;

49 as the active site sequence;

or contains CGPC SEQ ID NO;

48 as the active site sequence.

6. The method of claim 1 wherein the host is deficient in any one or more of thioredoxin reductase (trx8), glutathione reductase (gor), ompT or Ion gene products.

7. The method of claim 1 wherein a sequence encoding a cleavage site is located between the sequence encoding thioredoxin and the sequence encoding the eukaryotic protein.

8. The method of claim 7 wherein the cleavage site is susceptible to proteolysis.

9. The method of claim 8 wherein the cleavage site is a TEV cleavage site having a sequence selected from the group consisting of ENLYFQG (SEQ ID NO:

53) or ENLYFQS (SEQ ID NO;

     54).

10. The method of claim 7 wherein the cleavage site is susceptible to chemical cleavage.

11. The method of claim 10 wherein the cleavage site sequence has amino acid sequence DP.

12. The method of claim 1 wherein the fusion protein comprises a peptide sequence which is a ligand for a receptor.

13. The method of claim 12 wherein the peptide sequence comprises a poly histidine sequence.

14. The method of claim 1 wherein the host is a bacterium.

15. The method of claim 14 wherein the bacterium is E. coli.

16. The method of claim 15 wherein the E. coli is the Origami strain.

17. The method of claim 1 wherein the eukaryotic protein is a disintegrin.

18. The method of claim 17 wherein the disintegrin is a snake venom toxin selected from the group consisting of PI, PII or III class contortrostatin.

19. The method of claim 17 wherein the disintegrin comprises at its C-terminus a sequence encoding a binding domain for an integrin.

20. The method of claim 19 wherein said integrin is selected from the group consisting of integrins α

IIbβ

3, α

vβ

3, α

vβ

5, and α

5β

1.

21. The method of claim 19 wherein said sequence is HKGPAT (SEQ ID NO:

47).

22. The method of claim 1 wherein the eukaryotic protein comprises a disintegrin domain.

23. The method of claim 1 wherein the eukaryotic protein is selected from the group consisting of a cytokine, chemokine, interferon, growth factor, immunoglobulin, toxin and plasminogen activator.

24. The method of claim 1 wherein the eukaryotic protein is selected from the group consisting of jararhagin, disintegrin schistatin, snake metalloproteinase fibrolase, human interleukin-2 precursor, human interferon-γ

, human transforming growth factor beta 2, human liver expression chemokine, CCL16, omega-conotoxin CVID precursor, scorpion chlorotoxin human ADAM 9 precursor, human vascular endothelial factor A (VEGF-A), and human tissue-type plasminogen activator precursor (t-PA).

25. The method of claim 1 wherein the eukaryotic protein is a multimer or a monomer.

26. A method of expressing in prokaryotic host cells a disintegrin, said method comprising:

a) transforming a prokaryotic host cell with an expression vector encoding a fusion protein which comprises an N-terminal segment encoding thioredoxin and a C-terminal segment encoding a disintegrin; and

b) expressing said eukaryotic protein fused to said disintegrin,

wherein the host cell has been modified to express a disulfide isomerase DsbC in the cytoplasm of the cell, and

wherein the DsbC has the amino acid sequence as set forth in SEQ ID NO;

3 which is modified so that its active site sequence between positions 98-101 has been changed to CGFC (SEQ ID NO;

     45) or CTFC (SEQ ID NO;

     46).

27. The method of claim 26 wherein the host cell has been modified to express a thiol disulfide interchange protein (DsbD) alpha domain fragment in the cytoplasm.

28. The method of claim 27 wherein the DsbD alpha domain fragment consist of the amino acid sequence as set forth in SEQ ID NO:

4.

29. The method of claim 26 wherein the thioredoxin portion of the fusion protein has the amino acid sequence as set forth in SEQ ID NO:

1.

30. The method of claim 26 wherein the host is deficient in any one or more of thioredoxin reductase (trxB), glutathione reductase (gor), ompT or Ion gene products.

31. The method of claim 26 wherein a sequence encoding a cleavage site is located between the sequence encoding thioredoxin and the sequence encoding the eukaryotic protein.

32. The method of claim 26 wherein the fusion protein further comprises a peptide sequence which is a ligand for a receptor.

33. The method of claim 26 wherein the disintegrin is a snake venom toxin selected from the group consisting of PI, PII or III class or contortrostatin.

34. The method of claim 33 wherein the disintegrin comprises at its C-terminus a sequence encoding a binding domain for an integrin.

35. The method of claim 34 wherein said sequence is HKGPAT (SEQ ID NO:

47).

36. The method of claim 26 wherein the eukaryotic protein comprises a disintegrin domain.

37. The method of claim 26 wherein the eukaryotic protein is expressed as a monomer.

38. The method of claim 26 wherein the fusion protein is expressed at a level of at least 1-3 mg/L.

39. The method of claim 26 wherein the fusion protein is expressed at a level of at least 7-9 mg/L.