Patent ID: 6469145
Filing Date: 2002-10-22
Classification: C12N

Abstract:
A process for isolating organophosphorus hydrolase enzyme, comprising the steps of:(a) cultivating recombinant bacteria that express organophosphorus hydrolase enzyme in soluble form, said bacteria comprising Escherichia coli containing plasmid pVSEOP7; (b) lysing said cultivated recombinant bacteria into a buffered aqueous solution and separating out unlysed cells and cellular debris to provide an aqueous solution of cell free bacterial proteins; (c) contacting said aqueous solution of cell free bacterial proteins with a strong cation exchange resin, said aqueous solution comprising soluble organophosphorus hydrolase enzyme; (d) washing said strong cation exchange resin with a washing buffer to remove unbound proteins from said strong cation exchange resin; (e) eluting proteins that remain bound to said strong cation exchange resin by washing said resin with an eluting buffer comprising salt in a concentration that starts at about 0.0 M and is raised during the eluting process to about 0.5 M, and detecting and collecting eluate comprising a protein having organophosphorus hydrolase enzyme activity; and wherein steps (c), (d) and (e) are conducted in a single chromatography column at a pH that is less than the isoelectric point of the organophosphorus hydrolase enzyme, and wherein said organophosphorus hydrolase enzyme is recovered in a purity of at least about 90% relative to the total protein in the final product.