Process for efficiently producing transglutaminase through DNA recombination

• US 5,827,712 A
• Filed: 05/17/1996
• Issued: 10/27/1998
• Est. Priority Date: 05/17/1995
• Status: Expired due to Term

• Current Assignee: Ajinomoto Co., Inc.
• Original Assignee: Ajinomoto Co., Inc.
• Inventors: Yokoyama, Keiichi, Kikuchi, Yoshimi, Yasueda, Hisashi
• Primary Examiner(s): Grimes, Eric
• Assistant Examiner(s): Slobodyansky, Elizabeth
• Application Number:
  US08/649,193
• Time in Patent Office: 893 Days
• Field of Search: 435/69.1, 435/252.33, 435/193, 435/320.1, 536/23.2, 536/23.7, 530/350, 530/825
• US Class Current: 435/193
• CPC Class Codes: C07K 14/245 Escherichia (G)
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12N 9/1044 Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
• CPC Class Codes: Y10S 530/825 Bacteria

Abstract

The present invention relates to a process for producing a transglutaminase, which comprises incubating Escherichia coli expressing genes encoding a heat shock protein (DnaJ) and a transglutaminase. The transglutaminase is produced in large quantities, at low cost and has the appropriate stereostructure to render the transglutaminase biologically active. The transglutaminase so produced is useful in the food industry.

Claims

1. A process for producing a transglutaminase, which comprises incubating E. coli comprising a gene encoding a heat shock protein DnaJ and a gene encoding a transglutaminase, wherein each of said genes is operably linked in proper reading frame to sequences which direct the expression of each gene such that expression of the DnaJ gene is enhanced and the transglutaminase produced is biologically active and is present in soluble form at a level higher than that of transglutaminase expressed in E. coli in which expression of the DnaJ gene is not enhanced, and collecting the transglutaminase from said E. coli.

2. The process for producing a transglutaminase according to claim 1, wherein said E. coli is transformed with a vector comprising the DnaJ and transglutaminase genes operably linked in proper reading frame to sequences which direct the expression of said genes.

3. The process for producing a transglutaminase according to claim 1, wherein the strain of E. coli is a strain which over-expresses an endogenous gene encoding a heat shock protein DnaJ.

4. The process for producing a transglutaminase according to claim 3, wherein the E. coli is transformed with a vector comprising a gene encoding the transglutaminase, wherein said gene is operably linked in proper reading frame to sequences which direct the expression of said gene encoding the transglutaminase.

5. The process of claim 1, wherein the transglutaminase is a transglutaminase derived from fish.

6. The process of claim 5, herein the transglutaminase is a transglutaminase derived from a red sea bream.

7. The process of claim 1, wherein the E. coli further comprises a gene encoding DnaK.

8. The process for producing a transglutaminase according to claim 7, wherein said E. coli is transformed with a vector comprising the genes encoding the heat shock proteins DnaJ and DnaK and the gene encoding the transglutaminase operably linked in proper reading frame to sequences which direct the expression of said genes, such that the transglutaminase produced is biologically active and is present in soluble form at a level higher than that of transglutaminase expressed in E. coli in the absence of the gene encoding a heat shock protein DnaJ.

9. The process for producing a transglutaminase according to claim 7, wherein the heat shock proteins DnaJ and DnaK are over-expressed.

10. The process for producing a transglutaminase according to claim 9, wherein the strain of E. coli is a strain which over-expresses an endogenous gene encoding a heat shock protein DnaJ.

11. The process for producing a transglutaminase according to claim 10, wherein the E. coli is transformed with a vector comprising a gene encoding the transglutaminase, wherein said gene is operably linked in proper reading frame to sequences which direct the expression of said gene encoding the transglutaminase.

12. The process of claim 11, wherein the transglutaminase is a transglutaminase derived from fish.

13. The process of claim 12, wherein the transglutaminase is a transglutaminase derived from a red sea bream.

14. The process of claim 1, wherein the gene encoding the transglutaminase is present on plasmid pTTG-2.

15. The process of claim 1, wherein the gene encoding the DnaJ is present on plasmid pDnaJ-01.

16. The process of claim 7, wherein the gene encoding the DnaK is present on plasmid pDnaK-01.

17. The process of claim 7, wherein the genes encoding DnaJ and DnaK are present on plasmid pDnaKJ-01.

18. The process of claim 1, wherein the E. coli is AJ-13097, deposited at NIBHT under Deposit No. FERM BP-5438.

19. The process of claim 7, wherein the E. coli is AJ-13096, deposited at NIBHT under Deposit No. FERM BP-5437.

20. An E. coli transformed with sequences encoding a non-E. coli transglutaminase and a heat shock protein DnaJ, wherein expression of said DnaJ is enhanced.

21. The transformed E. coli of claim 20, identified as FERM BP-5438.

22. An E. coli which naturally expresses an enhanced level of DnaJ transformed with sequences encoding a non-E. coli transglutaminase and a heat shock protein DnaK.

23. An E. coli which naturally expresses an enhanced level of DnaJ transformed with sequences encoding a transglutaminase and GroESL.

24. An E. coli transformed with sequences encoding a transglutaminase and heat shock proteins DnaJ and DnaK, wherein expression of DnaJ is enhanced.

25. The transformed E. coli of claim 24, identified as FERM BP-5437.