Expression and purification of CRM197 and related proteins
Inventors
OGANESYAN, Natalia • Lees, Andrew
Assignees
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Publication Number
US-10093704-B2
Publication Date
2018-10-09
Expiration Date
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.
Core Innovation
The invention relates to producing a CRM protein in a recombinant cell by using an expression vector to drive CRM coding sequence expression cytoplasmically and obtaining the expressed CRM protein as a soluble CRM protein. A recombinant cell is provided in which the activity of one or more disulfide reductase enzymes is reduced so that the redox status of the cytoplasm shifts to a more oxidative state as compared to an unmodified recombinant cell.
CRM protein cytoplasmic expression is induced from a promoter functionally linked to a CRM coding sequence, and the CRM protein expressed is isolated as soluble. The reduced disulfide reductase activity is provided in which one or more disulfide reductase enzymes comprise one or more of an oxidoreductase, a dihydrofolate reductase, a thioredoxin reductase, or a glutathione reductase, thereby supporting formation of a soluble CRM protein having native disulfide bonds.
The disclosed purification and characterization approach uses chromatography with heparin/heparin-like resins and related chromatography resins, and characterization is performed using HB-EGF binding by ELISA. The CRM protein produced is natively folded and demonstrates correct receptor-binding behavior based on receptor-binding comparisons.
Claims Coverage
The document includes three independent claims (clm-00001, clm-00009, clm-00024). Each claim centers on producing a soluble CRM protein by cytoplasmic expression in a recombinant cell having reduced disulfide reductase activity that shifts cytoplasmic redox, followed by isolating the expressed soluble CRM protein.
Reduced disulfide reductase activity to shift cytoplasmic redox state and cytoplasmic soluble CRM production
Providing a recombinant cell containing an expression vector in which the recombinant cell has 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; inducing the expression vector to produce CRM protein cytoplasmically; and isolating the CRM protein expressed, wherein the CRM protein expressed is soluble.
Reduced disulfide reductase enzyme activity with specified reductase enzyme types
Providing a recombinant cell containing an expression vector wherein the recombinant cell has reduced activity of one or more disulfide reductase enzymes, and 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.
Native disulfide bonds in soluble CRM expressed cytoplasmically under reduced disulfide reductase activity
Providing a recombinant cell containing an expression vector wherein the expression vector contains a promoter functionally linked to a CRM coding sequence, and 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.
Across the independent claims, the core inventive coverage is cytoplasmic production of soluble CRM protein in recombinant cells with reduced disulfide reductase activity that shifts cytoplasmic redox to a more oxidative state, with further coverage including specifying disulfide-reductase enzyme types and achieving soluble CRM protein comprising native disulfide bonds.
Stated Advantages
Produces a CRM protein that is soluble when expressed cytoplasmically.
Shifts the cytoplasmic redox status to a more oxidative state compared to an unmodified recombinant cell.
Produces a CRM protein comprising native disulfide bonds.
Improves solubility and supports natively folded behavior as characterized by HB-EGF binding and receptor-binding equivalence described in the document.
Documented Applications
Use as a vaccine carrier via conjugation to pneumococcal polysaccharides, with monitoring of binding after conjugation described in the document.
Conjugate vaccine immunogenicity in mice is documented in the document.
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