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

Author:
Swiatek, Wojciech, Sugajska, Ewa, Lankiewicz, Leszek, Hemmings, Brian A., Zolnierowicz, Stanislaw
Source:
European journal of biochemistry 2000 v.267 no.16 pp. 5209-5216
ISSN:
0014-2956
Subject:
Pichia pastoris, agarose, antibodies, chromatography, histidine, homologous recombination, humans, inhibitory concentration 50, leucine, methanol, methylation, okadaic acid, phosphoprotein phosphatase, phosphorus, phosphorylase, rabbits, recombinant proteins, skeletal muscle, yeasts
Abstract:
Methylotrophic yeast Pichia pastoris was used for a medium‐scale expression of structural (PR65/A) and catalytic (PP2Ac) subunits of human type 2A protein phosphatase (PP2A). Constructs encoding these subunits, which were designed to introduce eight histidines at their N‐termini, were introduced into 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+/nitrilotriacetate/agarose. In addition, chromatography on ω‐aminohexyl‐Sepharose was applied to purify recombinant (r)PR65/A. This purification scheme yielded approximately 5 mg and 100 µg of rPR65/A and rPP2Ac, respectively, from 1 L of the yeast culture. The specific activity of rPP2Ac measured with [32P]phosphorylase a[1.7 µmol·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 complex as demonstrated both by activity assays in the presence of protamine and by chromatography on protamine–agarose. In summary, P. pastoris provides a convenient heterologous system for the production of recombinant subunits of PP2A.
Agid:
73674