Expression of protein histidine phosphatase in Escherichia coli, purification, and determination of enzyme activity

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. © 2007 Humana Press Inc.