Expression, purification, and characterization of human osteoclastic protein-tyrosine phosphatase catalytic domain in Escherichia coli

Protein Expr Purif. 2015 Mar:107:7-12. doi: 10.1016/j.pep.2014.11.008. Epub 2014 Nov 21.

Abstract

Osteoclastic protein tyrosine phosphatase (PTP-oc) is a structurally unique transmembrane protein tyrosine phosphatase (PTP) that contains only a relatively small intracellular PTP catalytic domain, does not have an extracellular domain, and lacks a signal peptide proximal to the NH2 terminus. The present study reports the expression, purification, and characterization of the intracellular catalytic domain of PTP-oc (ΔPTP-oc). ΔPTP-oc was expressed in Escherichia coli cells as a fusion with a six-histidine tag and was purified via nickel affinity chromatography. When with para-nitrophenylphosphate (p-NPP) as a substrate, ΔPTP-oc exhibited classical Michaelis-Menten kinetics. Its responses to temperature and ionic strength were similar to those of other PTPs. The optimal pH value of ΔPTP-oc is approximately 7.0, unlike other PTPs, whose optimal pH values are approximately 5.0.

Keywords: Enzyme kinetics; Escherichia coli; Nickel affinity chromatography; Osteoclastic protein tyrosine phosphatase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Catalytic Domain
  • Enzyme Stability
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression*
  • Humans
  • Kinetics
  • Osteoclasts / chemistry
  • Osteoclasts / enzymology*
  • Protein Tyrosine Phosphatases / chemistry*
  • Protein Tyrosine Phosphatases / genetics
  • Protein Tyrosine Phosphatases / isolation & purification*
  • Protein Tyrosine Phosphatases / metabolism

Substances

  • Protein Tyrosine Phosphatases