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US 12,234,266 B2Granted
CRM197 protein expression
AssigneeObi Pharma, Inc.
InventorsI-Ming Cho, Chia-Hung Chiu, Lee-Cheng Liu
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Abstract
The present invention relates to a method for producing CRM197 recombinant protein in cells. The method comprises culturing a cell comprising an expression plasmid with a polynucleotide and inducing expression of the CRM197 protein.
Claims5
A method of producing a CRM197 protein, comprising culturing a cell comprising an expression plasmid having a polynucleotide, wherein the polynucleotide comprises:
- (a) a CRM197 nucleotide sequence comprising SEQ ID NO:
- 1, and
- (b) a secretion signal sequence encoding amino acids of SEQ ID NO:
- 2 at 5′-terminus of the CRM197 nucleotide sequence;
- and
- inducing expression of the CRM197 protein, wherein the induction is performed at pH 7.5-8.0 in an expression medium, and an expression temperature of the CRM197 protein is first incubating at 37° C. and then reducing to about 20-25° C.,
- wherein the expression plasmid comprises a phosphate regulated promoter, and the cell comprises E. coli,
- wherein the phosphate regulated promoter is a bacterial alkaline phosphatase A promoter;
- and the CRM197 protein is soluble, periplasmic, and folded and comprises two intra-disulfide bonds.
6 more elements
A expression plasmid, comprising
- a polynucleotide of comprising:
- (a) a CRM197 nucleotide sequence at least 90% identical to SEQ ID NO:
- 1, and
- (b) a secretion signal sequence encoding amino acids of SEQ ID NO:
- 2 at 5′-terminus of the CRM197 nucleotide sequence;
- and
- an inducible promoter to initiate an expression of the polynucleotide,
- wherein the inducible promoter comprises a phosphate regulated promoter,
- wherein the expression plasmid is deposited under ATCC Accession Number PTA-124609 on Nov. 28, 2017.
6 more elements
Dependencies are inferred from the claim text.