Expression and purification of CRM197 and related proteins
AssigneeFina BioSolutions, LLC
InventorsNatalia 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. 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.
Claims39
A method of producing a CRM protein comprising:
- providing a recombinant cell that contains an expression vector, wherein the recombinant cell has a reduced activity of one or more disulfide reductase enzymes that shifts the redox status of the cytoplasm to a more oxidative state as compared to an unmodified recombinant cell, and the expression vector contains a promoter functionally linked to a CRM coding sequence;
- inducing the expression vector to produce CRM protein cytoplasmically;
- and
- isolating the CRM protein expressed, wherein the CRM protein expressed is soluble.
A method of producing a CRM protein comprising:
- providing a recombinant cell that contains an expression vector, wherein the recombinant cell has a reduced activity of one or more disulfide reductase enzymes and the expression vector contains a promoter functionally linked to a CRM coding sequence, wherein the one or more disulfide reductase enzymes comprises one or more of an oxidoreductase, a dihydrofolate reductase, a thioredoxin reductase or a glutathione reductase;
- expressing CRM protein from the CRM coding sequence cytoplasmically;
- and isolating the CRM protein expressed, wherein the CRM protein expressed is soluble.
A method of producing a CRM protein comprising:
- providing a recombinant cell that contains an expression vector, wherein the expression vector contains a promoter functionally linked to a CRM coding sequence, wherein the recombinant cell has reduced activity of one or more disulfide reductase enzymes;
- expressing CRM protein from the CRM coding sequence cytoplasmically;
- and isolating the CRM protein expressed, wherein the CRM protein expressed is soluble and comprises native disulfide bonds.
Dependencies are inferred from the claim text.