Protocol

Protein Phosphatase Protocols

Volume 365 of the series Methods in Molecular Biology pp 247-260

Expression of Protein Histidine Phosphatase in Escherichia coli, Purification, and Determination of Enzyme Activity

  • Nicole BäumerAffiliated withInstitut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität
  • , Anette MäurerAffiliated withInstitut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität
  • , Josef KrieglsteinAffiliated withInstitut für Pharmakologie and Toxikologie, Philipps-Universität
  • , Susanne KlumppAffiliated withInstitut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität

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Abstract

A protein histidine phosphatase (PHP) from vertebrates was first identified in 2002. Here we describe the expression of that PHP in Escherichia coli and purification of the recombinant protein. In addition, a detailed protocol is provided describing determination of PHP activity in vitro. Proteins phosphorylated on histidine residues in general cannot be easily obtained. This also applies to the substrates of PHP. To circumvent that obstacle, assay conditions are introduced enabling scientists to study PHP activity using a substrate within crude homogenates of cells and tissues.

Key Words

Dephosphorylation E. coli expression phosphorylation protein histidine phosphatase purification