Overexpression, purification, and characterization of SHPTP1, a Src homology 2-containing protein-tyrosine-phosphatase

Proc Natl Acad Sci U S A. 1993 Feb 1;90(3):1092-6. doi: 10.1073/pnas.90.3.1092.

Abstract

A protein-tyrosine-phosphatase (PTPase; EC 3.1.3.48) containing two Src homology 2 (SH2) domains, SHPTP1, was previously identified in hematopoietic and epithelial cells. By placing the coding sequence of the PTPase behind a bacteriophage T7 promoter, we have overexpressed both the full-length enzyme and a truncated PTPase domain in Escherichia coli. In each case, the soluble enzyme was expressed at levels of 3-4% of total soluble E. coli protein. The recombinant proteins had molecular weights of 63,000 and 45,000 for the full-length protein and the truncated PTPase domain, respectively, as determined by SDS/PAGE. The recombinant enzymes dephosphorylated p-nitrophenyl phosphate, phosphotyrosine, and phosphotyrosyl peptides but not phosphoserine, phosphothreonine, or phosphoseryl peptides. The enzymes showed a strong dependence on pH and ionic strength for their activity, with pH optima of 5.5 and 6.3 for the full-length enzyme and the catalytic domain, respectively, and an optimal NaCl concentration of 250-300 mM. The recombinant PTPases had high Km values for p-nitrophenyl phosphate and exhibited non-Michaelis-Menten kinetics for phosphotyrosyl peptides.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Bacteriophage T7 / genetics
  • Base Sequence
  • Escherichia coli / genetics*
  • Genes, src / genetics
  • Humans
  • Hydrogen-Ion Concentration
  • Molecular Sequence Data
  • Promoter Regions, Genetic / genetics
  • Protein Tyrosine Phosphatases / biosynthesis*
  • Protein Tyrosine Phosphatases / drug effects
  • Protein Tyrosine Phosphatases / genetics
  • Recombinant Proteins / biosynthesis
  • Sequence Homology, Amino Acid
  • Sodium Chloride / pharmacology
  • Substrate Specificity

Substances

  • Recombinant Proteins
  • Sodium Chloride
  • Protein Tyrosine Phosphatases