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

B Szoor, S Gross, L Alphey*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

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 NH2-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 (NH4)(2)SO4-ethanol precipitation followed by Ni2+-agarose affinity chromatography and Mono Q anion-exchange chromatography. This purification scheme yielded approximately 80 mug 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. (C) 2001 Elsevier Science.

Original languageEnglish
Pages (from-to)213-218
Number of pages6
JournalArchives of biochemistry and biophysics
Volume396
Issue number2
Publication statusPublished - 15 Dec 2001

Keywords

  • protein phosphatases
  • PP1
  • Drosophila
  • Pichia pastoris
  • protein purification
  • ALCOHOL OXIDASE GENES
  • OKADAIC ACID
  • INHIBITOR-2
  • MELANOGASTER
  • PURIFICATION
  • CHAPERONE
  • MITOSIS

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