Process for producing polypeptide having disulfide bond
First Claim
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:
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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.
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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.
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Citations
18 Claims
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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Specification