Biochemical characterization of recombinant Drosophila type 1 serine/threonine protein phosphatase (PP1c) produced in Pichia pastoris.

The methylotrophic yeast Pichia pastoris was used to express Drosophila melanogaster type 1beta serine/threonine phosphoprotein phosphatase catalytic subunit (PP1beta9C). A construct encoding PP1beta9C with a short NH(2)-terminal fusion including six histidine residues was introduced into the X-33 and KM71H strains of P. pastoris by homologous recombination. Recombinant protein was purified from cell free extracts 24 h after methanol induction. PP1beta9C was purified to a specific activity of 12,077 mU/mg by a three-step purification method comprising (NH(4))(2)SO(4)-ethanol precipitation followed by Ni(2+)-agarose affinity chromatography and Mono Q anion-exchange chromatography. This purification scheme yielded approximately 80 microg of active, soluble PP1beta9C per 1 L of culture. In contrast to recombinant PP1beta9C overexpressed in bacteria, which differs from native PP1c in several biochemical criteria including the requirement for divalent cations, sensitivity to vanadate, and p-nitrophenyl phosphate (pNPP) phosphatase activity, recombinant PP1beta9C produced in P. pastoris has native-like properties. P. pastoris thus provides a reliable and convenient system for the production of active, native-like recombinant PP1beta9C.