EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF THE RECOMBINANT NAD-MALIC ENZYME FROM ASCARIS-SUUM

The cDNA encoding the 65-kDa subunit of malic enzyme from Ascaris suum was cloned into the bacterial expression vector pKK223-3 and overprod uced in Escherichia coli. A protein with a subunit molecular mass of 6 5,000 was expressed at a level of up to 3% of the total soluble protei n in JM109, as judged by SDS-PAGE. The enzyme was purified using colum n chromatography on phenyl-Sepharose followed by orange-A agarose. The purification procedure resulted in a 32-fold purification with an ove rall yield of 51%. The bacterially expressed enzyme exhibits kinetic c onstants identical to those measured for native A. suum NAD-malic enzy me. (C) 1997 Academic Press.