Process for producing polypeptide having disulfide bond

• US 7,037,684 B2
• Filed: 03/13/2001
• Issued: 05/02/2006
• Est. Priority Date: 03/14/2000
• Status: Expired due to Fees

• Current Assignee: Itoham Foods Incorporated (Itoham Yonekyu Holdings, Inc.), Shigezo Udaka
• Original Assignee: Itoham Foods Incorporated (Itoham Yonekyu Holdings, Inc.), Shigezo Udaka
• Inventors: Sato, Seiji, Oka, Shusaku, Kondo, Masaaki, Kudo, Toshiyuki, Higashikuni, Naohiko, Udaka, Shigezo
• Primary Examiner(s): Prouty, Rebecca E.
• Assistant Examiner(s): Ramirez, Delia M.
• Application Number:
  US10/221,677
• Publication Number:
  US 20040018596A1
• Time in Patent Office: 1,876 Days
• Field of Search: 435/232, 435/320.1, 435/252.3, 435/252.31, 435/69.1, 435/69.8, 435/69.7, 435/69.4, 536/23.2, 536/23.4, 536/23.5, 530/303
• US Class Current: 435/69.4
• CPC Class Codes: C07K 14/32 from Bacillus (G)
• CPC Class Codes: C07K 14/62 Insulins
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/035 containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
• 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: C12N 15/625 containing a sequence coding for a signal sequence
• CPC Class Codes: C12N 15/75 for Bacillus
• CPC Class Codes: C12N 9/90 Isomerases (5.)
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione

Abstract

The invention relates to a DNA that enables co-expression of a protein disulfide isomerase and a polypeptide having disulfide bonds. The invention further relates to a process of producing polypeptides using the DNA and a process of enhancing the efficiency of formation of correct disulfide bond in an expression system of a gene recombinant polypeptide having disulfide bonds.

Claims

1. A DNA which enables co-expression of a protein disulfide isomerase and a polypeptide having disulfide bonds,

comprising at least one promoter required for gene expression, two identical or different Shine-Dalgamo sequences (SD), two identical or different sequences encoding signal peptides of cell wall proteins (CWP) of a bacterium of the genus Bacillus (CWPsp), a gene encoding a polypeptide having disulfide bonds which is a fusion protein comprising human insulin, and a gene encoding protein disulfide isomerase (PDI) that are ligated to each other as represented by the following formula:

5′

-Promoter-SD-CWPsp-X₁-(Promoter)n-SD-CWPsp-X₂-3′

,

wherein X₁=fusion protein and X₂=PDI, or X₁=PDI and X₂=fusion protein, and n=0 or 1, wherein if n=1 then the first promoter and the second promoter are identical or different and wherein if n=0, there is only one promoter,

wherein the fusion protein comprising a human insulin is represented by the following formula;

MWPmp9-GSLQPR-B chain-RGHRP-Linker-PR-A chain,

wherein MWPmp9 represents 9 amino acids from the N terminus of a middle-wall mature protein of bacteria of the genus Bacillus;

GSLQPR, RGHRP and PR each represents amino acids 30–

35, amino acids 66–

70 and amino acids 34–

35 of SEQ ID NO;

24, A chain and B chain respectively represent the A chain and B chain of human insulin; and

Linker represents a linker consisting of any amino acids.

2. The DNA of claim 1, wherein the promoter is derived from a bacterium of the genus Bacillus.

3. The DNA of claim 2, wherein the promoter is derived from a gene encoding Bacillus CWP.

4. The DNA of claim 3, wherein CWP is a middle wall protein (MWP).

5. The DNA of claim 1, wherein the protein disulfide isomerase is derived from a human.

6. A vector which comprises the DNA according to claim 1.

7. A bacterium of the genus Bacillus which is transformed with the vector of claim 6.

8. The bacterium of claim 7, wherein the bacterium of the genus Bacillus is Bacillus brevis.

9. A process for producing a polypeptide having disulfide bonds, which comprises:

introducing the vector according to claim 6 into a bacterium of the genus Bacillus;

culturing the obtained transformed bacterium in a medium;

co-expressing a protein disulfide isomerase and the polypeptide having one or more disulfide bonds, thereby secreting together extracellularly; and

collecting the polypeptide having correct disulfide bonds formed by the action of the protein disulfide isomerase.

10. The process of claim 9, which further comprises the steps of:

generating a polypeptide having correct disulfide bonds as a fusion protein; and

treating the obtained fusion protein with protease to obtain the polypeptide.

11. The DNA of claim 5, wherein the promoter is derived from a bacterium of the genus Bacillus.

12. The DNA of claim 11, wherein the promoter is derived from a gene encoding Bacillus CWP.

13. The DNA of claim 12, wherein CWP is a middle wall protein (MWP).

14. A vector which comprises the DNA according to claim 5.

15. A bacterium of the genus Bacillus which is transformed with the vector of claim 14.

16. The bacterium of claim 15, wherein the bacterium of the genus Bacillus is Bacillus brevis.

17. A process for producing a polypeptide having disulfide bonds, which comprises:

introducing the vector according to claim 14 into a bacterium of the genus Bacillus;

culturing the obtained transformed bacterium in a medium;

co-expressing a protein disulfide isomerase and the polypeptide having one or more disulfide bonds, thereby secreting together extracellularly; and

collecting the polypeptide having correct disulfide bonds formed by the action of the protein disulfide isomerase.

18. The process of claim 17, which further comprises the steps of:

generating a polypeptide having correct disulfide bonds as a fusion protein; and

treating the obtained fusion protein with protease to obtain the polypeptide.