Fusion proteins containing glutathione-s-transferase

• US 5,654,176 A
• Filed: 09/16/1994
• Issued: 08/05/1997
• Est. Priority Date: 05/28/1987
• Status: Expired due to Term

• Current Assignee: EMD Millipore Corporation (Merck & Co., Inc.)
• Original Assignee: Zenyth Therapeutics Limited (CSL Limited)
• Inventors: Smith, Donald Bruce
• Primary Examiner(s): Patterson, Jr., Charles L.
• Assistant Examiner(s): GRIMES, ERIC BURTON
• Application Number:
  US08/307,337
• Time in Patent Office: 1,054 Days
• Field of Search: 435/69.7, 435/172.3, 435/320.1, 435/252.3, 435/69.1, 435/240.2, 435/252.33, 536/23.4, 530/350, 530/300
• US Class Current: 435/69.7
• CPC Class Codes: C07K 2319/23 containing a GST-tag
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins

Abstract

A recombinant DNA molecule comprising a nucleotide sequence which codes for expression of a fusion protein in which a foreign protein or peptide is fused with the enzyme glutathione-S-transferase, is disclosed, as well as expression vectors or host cells containing such a molecule. Optionally, the foreign protein or peptide is fused to the enzyme through a cleavable link. Also disclosed is an expression vector having inserted therein a nucleotide sequence capable of being expressed as the enzyme glutathione-S-transferase followed by at least one restriction endonuclease recognition site for insertion of a nucleotide sequence capable of being expressed as a foreign protein or peptide fused to the glutathione-S-transferase.

Claims

1. A recombinant DNA molecule comprising a nucleotide sequence which codes for a fusion protein wherein said fusion protein comprises glutathione-S-transferase and a second protein or peptide fused directly or indirectly with the COOH-terminus of glutathione-S-transferase, and wherein said fusion protein is capable of binding to glutathione.

2. The recombinant DNA molecule according to claim 1, wherein said glutathione-S-transferase is the 26kDa glutathione-S-transferase of Schistosoma japonicum.

3. The recombinant DNA molecule according to claim 1 wherein said second protein or peptide is fused to said glutathione-S-transferase through a cleavable link.

4. The recombinant DNA molecule according to claim 3, wherein said cleavable link is cleavable by a site specific protease.

5. The recombinant DNA molecule according to claim 4, wherein said clearable link is cleavable by thrombin, blood coagulation factor X_a or renin.

6. The recombinant DNA molecule of claim 1 wherein said glutathione-S-transferase is a mammalian glutathione-S-transferase.

7. An expression vector comprising the recombinant DNA molecule of any one of claims 1, 2-5 and 6.

8. A host cell comprising the expression vector of claim 7.

9. The host cell of claim 8 wherein said host cell is a bacterial cell.

10. The host cell according to claim 9 which is E. coli.

11. An expression vector comprising, in the 5'"'"' to 3'"'"' direction, a promoter, a nucleotide sequence encoding glutathione-S-transferase and a nucleotide sequence comprising at least one restriction endonuclease recognition site.

12. The expression vector according to claim 11, wherein said glutathione-S-transferase is the 26kDa glutathione-S-transferase of Schistosoma japonicum.

13. The expression vector according to claim 11, which comprises a plasmid selected from the group consisting of pGEX-1, pSj10 DBamI and pSj10 DBam7Stop7.

14. An expression vector comprising, in the 5'"'"' to 3'"'"' direction, a promoter, a nucleotide sequence encoding glutathione-S-transferase, a nucleotide sequence encoding a clearable link and a nucleotide sequence comprising at least one restriction endonuclease recognition site.

15. The expression vector according to claim 14, wherein said cleavable link is cleavable by a site specific protease.

16. The expression vector according to claim 14 which is pGEX-2T or pGEX-3X.

17. The plasmid pGEX-1.

18. The plasmid pGEX-2T.

19. The plasmid pGEX-3X.

20. A method of producing a fusion protein comprising glutathione-S-transferase and a second protein or peptide fused directly or indirectly with the COOH-terminus of glutathione-S-transferase which comprises:

(a) transforming a host cell with an expression vector comprising a promoter operatively linked to a nucleotide sequence which codes for a fusion protein wherein said fusion protein comprises glutathione-S-transferase and a second protein or peptide fused directly or indirectly with the COOH-terminus of glutathione-S-transferase and wherein said fusion protein is capable of binding to glutathione;

(b) culturing said host cell under conditions such that said fusion protein is expressed in recoverable quantity;

(c) lysing said host cell; and

(d) purifying said fusion protein by glutathione-affinity chromatography.

21. A method of producing a protein or peptide which comprises:

(a) transforming a host cell with an expression vector comprising a promoter operatively linked to a nucleotide sequence which codes for a fusion protein wherein said fusion protein comprises glutathione-S-transferase and a second protein or peptide fused with the COOH-terminus of glutathione-S-transferase through a cleavable link wherein said fusion protein is capable of binding to glutathione;

(b) culturing said host cell under conditions such that said fusion protein is expressed in recoverable quantity;

(c) lysing said host cell;

(d) purifying said fusion protein by glutathione-affinity chromatography;

(e) cleaving said protein or said peptide from said glutathione-S-transferase; and

(f) isolating said protein or said peptide.

22. A fusion protein comprising glutathione-S-transferase and a second protein or peptide fused directly or indirectly with the COOH-terminus of said glutathione-S-transferase wherein said fusion protein is capable of binding to glutathione.

23. A fusion protein comprising from amino to carboxyl terminus, glutathione-S-transferase, a cleavable linker and a second protein or peptide wherein said fusion protein is capable of binding to glutathione.

24. The fusion protein of claim 23 wherein said cleavable linker is cleavable by a site-specific protease.

25. The fusion protein of claim 24 wherein said protease is selected from the group consisting of thrombin, blood coagulation Factor Xa and renin.