Production of soluble recombinant proteins without N-terminal methionine in E-coli
AssigneeFina BioSolutions, LLC
InventorsMin-Ju Chang, Natalia Oganesyan, Andrew Lees
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Abstract
The present invention is directed to the cells, compositions and methods for the production of recombinant protein, wherein an f-met group on the 5′-terminus is enzymatically removed. In particular, the invention is directed to a production process for obtaining high levels of soluble recombinant CRM197 protein from E. coli. Cells preferably contain one or more mutations of disulfide reductase genes, so that disulfide reductase activity is reduced. The invention also relates to purification method for CRM197 as well as characterization of properly folded CRM197 protein.
Claims35
A method of producing a peptide comprising:
- expressing the peptide in a recombinant cell containing a gene that encodes a peptidase enzyme,
- wherein the gene that encodes the peptidase enzyme is integrated into the genome of the recombinant cell,
- wherein the recombinant cell has a reduced activity of one or more disulfide reductase enzymes,
- wherein the reduced activity of one or more disulfide reductase enzymes results in a shift in the redox status of the cytoplasm to a more oxidative state as compared to a recombinant cell that does not have reduced activity of one or more disulfide reductase enzymes, and
- wherein the peptide contains an N-terminal methionine;
- expressing the peptidase enzyme which removes the N-terminal methionine from the peptide;
- and
- isolating the peptide.
A method of producing a peptide containing disulfide bonds comprising:
- expressing the peptide in a recombinant cell containing a gene that encodes a peptidase enzyme,
- wherein the peptide is encoded in an expression vector,
- wherein the gene that encodes the peptidase enzyme is integrated into the genome of the recombinant cell,
- wherein the recombinant cell has a reduced activity of one or more disulfide reductase enzymes,
- wherein the recombinant cell is E. coli, and
- wherein the peptide contains an N-terminal methionine;
- expressing the peptidase enzyme which removes the N-terminal methionine from the peptide;
- and
- isolating the peptide from within the cytoplasm of the recombinant cell, wherein the peptide isolated is soluble.
A method of producing a protein comprising:
- providing an expression vector that encodes the protein, wherein the protein contains one or more sulfide linkages;
- expressing the expression vector in a recombinant cell that has a reduced activity of a disulfide reductase enzyme which results in a shift in the redox status of the cytoplasm of the recombinant cell to a more oxidative state as compared to a recombinant cell that does not have reduced activity of the disulfide reductase enzyme;
- and
- isolating the protein expressed by the recombinant cell wherein the protein expressed is correctly folded and soluble in the cytoplasm.
Dependencies are inferred from the claim text.