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.
Szöor
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B
Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS, United Kingdom.
Gross
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S
Alphey
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L
D000818:Animals; D001671:Biochemistry / Q000379:methods; D002852:Chromatography, Ion Exchange; D004330:Drosophila / Q000201:enzymology*; D004926:Escherichia coli / Q000378:metabolism; D000431:Ethanol / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000096:biosynthesis / Q000737:chemistry*; D010843:Pichia / Q000378:metabolism; D054645:Protein Phosphatase 1; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D011995:Recombination, Genetic; D013997:Time Factors
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D019319:Okadaic Acid; D000431:Ethanol; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1