Biochemical characterization of recombinant subunits of type 2A protein phosphatase overexpressed in Pichia pastoris

Methylotrophic yeast Pichia pastoris was used for a medium-scale expression of structural (PR65/A) and catalytic (PP2Ac) subunits of human type 2A pro tein phosphatase (PP2A). Constructs encoding these subunits, which were des igned to introduce eight histidines at their N-termini, were introduced int o the KM71 Pichia strain by homologous recombination. Recombinant proteins overproduced after methanol induction were purified from cell-free extracts by anion-exchange chromatography on DEAE-Sepharose, and Ni2+/nitrilotriace tate/agarose. In addition, chromatography on omega-aminohexyl-Sepharose was applied to purify recombinant (r)PR65/A. This purification scheme yielded approximately 5 mg and 100 mu g of rPR65/A and rPP2Ac, respectively, from 1 L of the yeast culture. The specific activity of rPP2Ac measured with [P-3 2]phosphorylase a [1.7 mu mol.min(-1).(mg protein)(-1)] and its inhibition by okadaic acid (IC50 = 0.66 nM) were similar to PP2A isolated from rabbit skeletal muscle. As demonstrated by immunodetection with methylation state- specific antibodies, recombinant PP2Ac was carboxymethylated at the last C- terminal leucine residue. Recombinant PP2A subunits were able to form a com plex as demonstrated both by activity assays in the presence of protamine a nd by chromatography on protamine-agarose. In summary, P. pastoris provides a convenient heterologous system for the production of recombinant subunit s of PP2A.