Process for producing polypeptide having disulfide bond

• US 20040018596A1
• Filed: 09/13/2002
• Published: 01/29/2004
• Est. Priority Date: 03/14/2000
• Status: Active Grant

• 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, Kondo, Masaaki, Kudo, Toshiyuki, Oka, Shusaku, Higashikuni, Naohiko, Udaka, Shigezo
• Granted Patent: US 7,037,684 B2
• Time in Patent Office: Days
• US Class Current: 435/69.7
• 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 present invention relates to a DNA which enables co-expression of a protein disulfide isomerase and a polypeptide having disulfide bonds,

comprising one or two identical or different promoters required for gene expression (Promoter), two identical or different Shine-Dalgarno 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 (Polypeptide), 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=Polypeptide and X₂=PDI, or X₁=PDI and X₂=Polypeptide, and n=0 or 1.

The present invention relates to also a process for producing polypeptides using the DNA. The present invention can enhance 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 one or two identical or different promoters required for gene expression (Promoter), two identical or different Shine-Dalgarno 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 (Polypeptide), 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=Polypeptide and X₂=PDI, or X₁=PD₁ and X₂=Polypeptide, and n=0 or 1.

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 CWP of a bacterium of the genus Bacillus.

4. The DNA of claim 3, wherein CWP is MWP.

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

6. The DNA of claim 1, wherein the polypeptide having disulfide bonds is a fusion protein containing a human insulin.

7. The DNA of claim 6, wherein the fusion protein containing 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 an amino acid sequence in an one-letter code;

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.

8. A vector which comprises the DNA of any one of claims 1 to 7.

9. A bacterium of the genus Bacillus which is transformed with the vector of claim 8.

10. The bacterium of claim 9, wherein the bacterium of the genus Bacillus is Bacillus brevis.

11. A process for producing a polypeptide having disulfide bonds, which comprises the steps of:

introducing the vector of claim 8 or 9 into ab acterium 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.

12. The process of claim 11, 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.

13. The process of claim 11 or 12, wherein the polypeptide is a human insulin.