Functional expression, purification, and characterization of recombinant human pyruvate carboxylase

The cDNA-encoding human pyruvate carboxylase (hPC) has been assembled and c loned into a very high efficiency mammalian expression vector and the const ruct transfected into 293T kidney cells. Stable clones expressing very high levels of hPC were produced and used as a source of the enzyme. Purificati on of the recombinant hPC was performed by selective precipitation with 40% ammonium sulfate followed by a single step avidin affinity chromatography, with an overall yield of 20%. Recombinant hPC purified by this method yiel ded a single band on SDS-PAGE with a specific activity of 20 U/mg. Kinetic analysis demonstrated that the recombinant human PC has the same properties as the native enzyme isolated from liver autopsy. This is the first report of production and purification of recombinant PC. (C) 1999 Academic Press.