Expression, Purification, and Kinetic Analysis of PTP Domains

Methods Mol Biol. 2016;1447:39-66. doi: 10.1007/978-1-4939-3746-2_3.

Abstract

Protein tyrosine phosphatases (PTP) are a large group of enzymes which work together with protein tyrosine kinases to control the tyrosine phosphorylation of proteins, thus playing a major role in cellular signaling. Here, we provide detailed protocols for expression and purification of the catalytic domain of RPTPμ and full length Eya3 as well as the extracellular region of PTPBR7. Methods are described for evaluation of the purity of the recombinant proteins thus obtained. For the purified Eya3 phosphatase we provide protocols for enzyme activity assay using either chromogenic, fluorescent, or peptide substrates. Determination of kinetic parameters by different graphical and computer-based procedures is also described.

Keywords: 6×His and GST tagged recombinant proteins; DiFMUP; Eukaryotic expression; PTP domains; PTP kinetics; PTP purification; Phosphopeptide; Prokaryotic expression; pNPP.

MeSH terms

  • Blotting, Western / methods
  • Catalytic Domain
  • Chromatography, Affinity / methods
  • Chromatography, Gel / methods
  • Chromatography, Ion Exchange / methods
  • Electrophoresis, Polyacrylamide Gel / methods
  • Escherichia coli / genetics
  • Gene Expression
  • Humans
  • Kinetics
  • Mass Spectrometry / methods
  • Protein Tyrosine Phosphatases / chemistry*
  • Protein Tyrosine Phosphatases / genetics
  • Protein Tyrosine Phosphatases / isolation & purification
  • Protein Tyrosine Phosphatases / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism

Substances

  • Recombinant Proteins
  • Protein Tyrosine Phosphatases