Expression of fusion polypeptides transported out of the cytoplasm without leader sequences

• US 5,914,254 A
• Filed: 05/12/1997
• Issued: 06/22/1999
• Est. Priority Date: 08/02/1993
• Status: Expired due to Fees

• Current Assignee: Celtrix Pharmaceuticals, Inc. (Insmed Incorporated)
• Original Assignee: Celtrix Pharmaceuticals, Inc. (Insmed Incorporated)
• Inventors: Zhang, Yang, Nguyen, Kimyen B., Cohen, Pedro A., Olsen, David R., Mascarenhas, Desmond, Olson, Pamela S.
• Primary Examiner(s): ULM, JOHN D
• Application Number:
  US08/854,811
• Time in Patent Office: 771 Days
• Field of Search: 435/69.7, 435/172.3, 435/252.3, 435/320.1, 530/350, 536/23.4
• US Class Current: 435/69.7
• CPC Class Codes: C07K 14/4743 Insulin-like growth factor binding protein
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/32 fusions with soluble part of a cell surface receptor, "decoy receptors"
• CPC Class Codes: C07K 2319/35 containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
• CPC Class Codes: C07K 2319/75 containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
• CPC Class Codes: C07K 2319/95 containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)

Abstract

The invention is directed to the use of a fusion partner that does not contain a leader sequence, as a means to increase the solubility and activity of recombinant polypeptides by facilitating the expression of fusion proteins, which are then transported out of the cytoplasm. The invention includes a nucleic acid encoding a fusion polypeptide comprising a mature interleukin-1-like polypeptide or a leader-deleted-translocating polypeptide, and a polypeptide of interest; as well as host cells comprising such nucleic acids, and fusion proteins so encoded. The invention also encompasses methods of using such nucleic acids to produce recombinant fusion polypeptides, mature polypeptides of interest, and purified compositions thereof.

Claims

1. A nucleic acid encoding a fusion polypeptide, said fusion polypeptide comprising:

(a) a fusion partner comprising a polypeptide selected from the group consisting of IL-1-α

, acidic FGF and basic FGF, int-2, hst/KS3, FGF-5, FGF-6, keratinocyte growth factor (KGF);

hisactophilin;

soybean trypsin inhibitor, Vibrio cholerae TcpG, leaderless DsbA and leaderless DsbC;

(b) a linker peptide; and

(c) mutant insulin-like growth factor binding protein 3 (IGFBP-3) wherein said linker peptide is positioned between said fusion partner and the IGFBP-3 and wherein said fusion partner constitutes the amino terminus of the fusion protein.

2. The nucleic acid of claim 1, wherein the fusion partner is selected from the group consisting of leaderless DsbA and leaderless DsbC.

3. The nucleic acid of claim 2 wherein said fusion partner lacks a double cysteine active site loop domain.

4. The nucleic acid of claim 1 wherein said fusion partner is leaderless DsbA lacking a double cysteine active site loop domain.

5. An expression vector comprising the nucleic acid of claim 4.

6. A host cell comprising the nucleic acid of claim 4.

7. An expression vector comprising the nucleic acid of claim 1.

8. A host cell comprising the nucleic acid of claim 1.

9. A host cell comprising:

a) an expression vector capable of expressing in said host cell a fusion polypeptide, said fusion polypeptide comprising,

i) a fusion partner, said fusion partner comprising a polypeptide selected from the group consisting of IL-1-α

, acidic FGF and basic FGF, int-2, hst/KS3, FGF-5, FGF-6, keratinocyte growth factor (KGF);

hisactophilin;

soybean trypsin inhibitor, Vibrio cholerae TcpG, leaderless DsbA and leaderless DsbC;

ii) a linker peptide comprising a cleavage site; and

iii) mutant insulin-like growth factor binding protein 3 (IGFBP-3), wherein said linker peptide is positioned between said fusion partner and the IGFBP-3 and wherein said fusion partner constitutes the amino terminus of the fusion protein; and

b) a nucleic acid capable of expressing in said host cell a proteolytic enzyme that specifically recognizes said cleavage site.

10. The host cell of claim 9 wherein said fusion partner is leaderless DsbA.

11. The host cell of claim 10 wherein said leaderless DsbA is lacking a double cysteine active site loop domain.

12. A fusion polypeptide comprising:

(a) a fusion partner comprising a polypeptide selected from the group consisting of IL-1-α

, acidic FGF and basic FGF, int-2, hst/KS3, FGF-5, FGF-6, keratinocyte growth factor (KGF);

hisactophilin;

soybean trypsin inhibitor, Vibrio cholerae TcpG, leaderless DsbA, and leaderless DsbC;

(b) a linker peptide; and

(c) mutant insulin-like growth factor binding protein 3 (IGFBP-3), wherein said linker peptide is positioned between said fusion partner and the IGFBP-3 and wherein said fusion partner constitutes the amino terminus of the fusion protein.

13. The fusion polypeptide of claim 12 wherein said fusion partner is selected from the group consisting of leaderless DsbA and leaderless DsbC.

14. The fusion polypeptide of claim 13 wherein said fusion partner lacks a double cysteine active site loop domain.

15. The fusion polypeptide of claim 12 wherein the fusion partner is a leaderless DsbA lacking a double cysteine active site loop domain.

16. The fusion polypeptide of claim 12 wherein said linker peptide comprises a cleavage site.

17. The fusion polypeptide of claim 16 wherein said cleavage site is a ubiquitin molecule.

18. A method of producing a substantially purified fusion polypeptide encoded by a nucleic acid, wherein said fusion polypeptide comprises,

(i) a fusion partner comprising a polypeptide selected from the group consisting of IL-1-α

, acidic FGF and basic FGF, int-2, hst/KS3, FGF-5, FGF-6, keratinocyte growth factor (KGF);

hisactophilin;

soybean trypsin inhibitor, Vibrio cholerae TcpG, leaderless DsbA and leaderless DsbC;

(ii) a linker peptide; and

(iii) mutant insulin-like growth factor binding protein 3 (IGFBP-3), wherein said linker peptide is positioned between said fusion partner and the IGFBP-3 and wherein said fusion partner constitutes the amino terminus of the fusion polypeptide;

said method comprising the steps of;

(a) introducing said nucleic acid encoding said fusion polypeptide into a host cell, thereby producing a transformed host cell;

(b) culturing said transformed host cell under conditions appropriate for expressing said fusion polypeptide; and

(c) purifying said fusion polypeptide, thereby obtaining a substantially purified fusion polypeptide.

19. The method of claim 18 wherein said fusion partner is leaderless DsbA.

20. The method of claim 18, wherein said fusion partner is leaderless DsbA lacking a double-cysteine loop active site domain.

21. A method of producing a substantially purified polypeptide of interest, said method comprising the steps of:

(a) introducing into a host cell a nucleic acid encoding a fusion polypeptide, said fusion polypeptide comprising,

(i) a fusion partner comprising a polypeptide selected from the group consisting of IL-1-α

, acidic FGF and basic FGF, int-2, hst/KS3, FGF-5, FGF-6, keratinocyte growth factor (KGF);

hisactophilin;

soybean trypsin inhibitor, Vibrio cholerae TcpG, leaderless DsbA and leaderless DsbC;

(ii) a linker peptide encoding a cleavage site, wherein said linker peptide is positioned between said fusion partner and said polypeptide of interest; and

(iii) mutant insulin-like growth factor binding protein 3 (IGFBP-3), wherein said linker peptide is positioned between said fusion protein and the IGFBP-3 and wherein said fusion partner constitutes the amino terminus of the fusion polypeptide, thereby producing a transformed host cell;

(b) culturing said transformed host cell under conditions appropriate for expressing said fusion polypeptide, thereby expressing said fusion polypeptide;

(c) cleaving said fusion polypeptide with a proteolytic enzyme or cleavage agent that recognizes said proteolytic cleavage site, thereby producing said mutant IGFBP-3; and

(d) purifying said mutant IGFBP-3, thereby obtaining a substantially purified mutant IGFBP-3.

22. The method of claim 21 wherein said fusion partner is leader less DsbA.

23. The method of claim 21, wherein said fusion partner is leaderless DsbA lacking a double-cysteine loop active site domain.

24. A method of producing a substantially purified polypeptide of interest, said method comprising the steps of:

(a) introducing into a host cell a nucleic acid encoding a fusion polypeptide, said fusion polypeptide comprising,

(i) a fusion partner comprising a polypeptide selected from the group consisting of IL-1-α

, acidic FGF and basic FGF, int-2, hst/KS3, FGF-5, FGF-6, keratinocyte growth factor (KGF);

hisactophilin;

soybean trypsin inhibitor, Vibrio cholerae TcpG, leaderless DsbA and leaderless DsbC;

(ii) a linker peptide encoding a cleavage site, wherein said linker peptide is positioned between said fusion partner and said polypeptide of interest; and

(iii) mutant insulin-like growth factor binding protein 3 (IGFBP-3), wherein said linker peptide is positioned between said fusion partner and the IGFBP-3 and wherein said fusion partner constitutes the amino terminus of the fusion protein;

thereby producing a transformed host cell;

(b) culturing said transformed host cell under conditions appropriate for expressing said fusion polypeptide, thereby expressing said fusion polypeptide;

(c) purifying said fusion polypeptide, thereby producing a substantially purified fusion polypeptide;

(d) cleaving said substantially purified fusion polypeptide with a proteolytic enzyme or cleavage agent that recognizes said proteolytic cleavage site, thereby producing said mutant IGFBP-3; and

(e) purifying said mutant IGFBP-3, thereby obtaining a substantially purified mutant IGFBP-3.

25. The method of claim 24 wherein said fusion partner is leaderless DsbA.

26. The method of claim 25 wherein said leaderless DsbA is lacking a double cysteine active site loop domain.

27. A method of producing a substantially purified polypeptide of interest comprising the steps of:

(a) introducing into a host cell a nucleic acid encoding a fusion polypeptide, said fusion polypeptide comprising,

(i) a fusion partner comprising a polypeptide selected from the group consisting of IL-1-α

, acidic FGF and basic FGF, int-2, hst/KS3, FGF-5, FGF-6, keratinocyte growth factor (KGF);

hisactophilin;

soybean trypsin inhibitor, Vibrio cholerae TcpG, leaderless DsbA and leaderless DsbC;

(ii) a linker peptide encoding a cleavage site, wherein said linker peptide is positioned between said fusion partner and said polypeptide of interest; and

(iii) mutant insulin-like growth factor binding protein 3 (IGFBP-3), wherein said linker peptide is positioned between said fusion partner and the IGFBP-3 and wherein said fusion partner constitutes the amino terminus of the fusion polypeptide;

and further wherein said host cell comprises a nucleic acid capable of expressing in said host cell a proteolytic enzyme that specifically recognizes said cleavage site;

thereby producing a transformed host cell;

(b) culturing said transformed host cell under conditions appropriate for expressing said fusion polypeptide and said proteolytic enzyme, thereby expressing said fusion polypeptide, causing the in vivo cleavage of said fusion polypeptide, and producing said mutant IGFBP-3; and

(c) purifying said polypeptide of interest, thereby obtaining a substantially purified mutant IGFBP-3.

28. The method of claim 27 wherein said fusion partner is leaderless DsbA.

29. The method of claim 27, wherein said leaderless DsbA is lacking a double-cysteine active site loop domain.