Process for the recovery of recombinantly produced chymosin from insoluble aggregate
AssigneeCelltech Limited
InventorsNorman H. Carey, Michael T. Doel, Timothy J. R. Harris, Peter A. Lowe
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Abstract
The production of recombinant chymosin is disclosed in which an insoluble form of chymosin precursor is produced by a bacterial host cell transformed by a vector including a coding sequence for said precursor. Solubilization of said insoluble form of chymosin precursor is accomplished using urea at a concentration of at least 7M or guanidine hydrochloride at a concentration of at least 6M prior to cleaving said precursor to form chymosin. Said solubilization preferably additionally involves the denaturation of said precursor in an alkaline aqueous solution, e.g., at a pH between a 9 and 11.5
Claims10
In the process for the production of bovine chymosin comprising the steps of producing an insoluble aggregate form of the chymosin precursor preprochymosin, methionine-prochymosin or methionine-chymosin from a bacterial host cell transformed with a vector including a coding sequence for said chymosin precursor, solubilizing said insoluble aggregate form of said chymosin precursor to produce a soluble native form of said chymosin precursor to produce bovine chymosin, the improvement wherein said solubilizing is carried out by a combination of:
- 1) denaturing said insoluble aggregate form of said chymosin precursor in an aqueous solution comprising urea at a concentration of at least 7M, and
- 2) denaturing said insoluble aggregate form of said chymosin precursor in an aqueous solution of alkali hydroxide between pH 10 and pH 11.5, and, subsequent to steps (1) and (2), reducing the concentration of urea and reducing the pH of the solution below a pH effective to denature said chymosin precursor to produce the soluble native form thereof.
Dependencies are inferred from the claim text.