DsbA/DsbB/DsbC/DsbD expression plasmid
AssigneeHSP Research Institute, Inc.
InventorsYoichi Kurokawa, Hideki Yanagi, Takashi Yura
Life of this patent
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Abstract
An artificial operon comprising polynucleotides encoding each of DsbA, DsbB, DsbC and DsbD; an expression plasmid carrying the above artificial operon, usable for expression of DsbA, DsbB, DsbC and DsbD; a cotransformant harboring the above expression plasmid and an expression vector for a foreign protein; and a method for producing a foreign protein comprising culturing the cotransformant.
Claims14
An artificial operon comprising:
- (a) a polynucleotide comprising a nucleic acid sequence which encodes a polypeptide (DsbA) comprising an amino acid sequence set forth in SEQ ID NO:1, or
- (b) a polynucleotide comprising a nucleic acid sequence encoding DsbA that hybridizes under stringent conditions to the complement of SEQ ID NO:1,
- wherein the polypeptide encoded by the polynucleotide forms disulfide bonds in a nascent polypeptide chain which has been transferred into the periplasm;
- (c) a polynucleotide comprising a nucleic acid sequence which encodes a polypeptide (DsbB) comprising an amino acid sequence set forth in SEQ ID NO:3, or
- (d) a polynucleotide comprising a nucleic acid sequence encoding DsbB that hybridizes under stringent conditions to the complement of SEQ ID NO:3,
- wherein the polypeptide encoded by the polynucleotide reoxidizes DsbA;
- (e) a polynucleotide comprising a nucleic acid sequence which encodes a polypeptide (DsbC) comprising an amino acid sequence set forth in SEQ ID NO:5, or
- (f) a polynucleotide comprising a nucleic acid sequence encoding DsbC that hybridizes under stringent conditions to the complement of SEQ ID NO:5,
- wherein the polypeptide encoded by the polynucleotide corrects the disulfide by means of cleavage of the disulfide bonds followed by re-crosslinking;
- and
- (g) a polynucleotide comprising a nucleic acid sequence which encodes a polypeptide (DsbD) comprising an amino acid sequence set forth in SEQ ID NO:7, or
- (h) a polynucleotide comprising a nucleic acid sequence encoding DsbD that hybridizes under stringent conditions to the complement of SEQ ID NO:7,
- wherein the polypeptide encoded by the polynucleotide reduces DsbC.
An artificial operon comprising a polynucleotide encoding DsbC and a polynucleotide encoding DsbD, wherein the polynucleotide encoding DsbC is selected from the group consisting of
- (e) a polynucleotide comprising a nucleic acid sequence which encodes a polypeptide comprising an amino acid sequence of SEQ ID NO:
- 5 (DsbC), or
- (f) a polynucleotide comprising a nucleic acid sequence that specifically hybridizes under stringent conditions to the complement of (e),
- wherein the polypeptide encoded by the polynucleotide corrects the disulfide by means of cleavage of the disulfide bonds followed by re-crosslinking;
- and the polynucleotide encoding DsbD is selected from the group consisting of
- (g) a polynucleotide comprising a nucleotide sequence which encodes a polypeptide comprising an amino acid sequence of SEQ ID NO:7 (DsbD), or
- (h) a polynucleotide comprising a nucleic acid sequence that specifically hybridizes to the complement of (g),
- wherein the polypeptide encoded by the polynucleotide re-reduces DsbC.
Dependencies are inferred from the claim text.