Chapter 37 Expression and Assay of Autophosphorylation of Recombinant Protein Kinases

Abstract

Publisher Summary This chapter outlines the various factors that must be considered while designing an experimental system to study recombinant protein kinases. The expression of recombinant protein kinases in Escherichia coli is a simple and inexpensive way to produce milligram amounts of an active protein kinase from a cloned gene. These recombinant enzymes are useful tools to examine the biochemical properties of any cloned protein kinase. To facilitate purification, fusion proteins are constructed using commercially available expression vectors. These fusion proteins contain a protein tag that allows that recombinant enzyme to be purified by affinity chromatography. Fusion proteins are not only easy to purify but the addition of a well-characterized protein tag often helps the solubility and stability of the recombinant protein. To further enhance the recovery of these recombinant enzymes host strains that are protease deficient and growth conditions that favor the recovery of soluble fusion protein are used. To ensure that the catalytic properties of the expressed proteins are not unduly influenced by the affinity tag or its purification, the activities of two different recombinant fusion proteins that have the same protein kinase catalytic domains are compared. These analyses demonstrate that the protein kinase fusions have essentially identical enzymatic properties. The availability of large amounts of protein kinase allows determining the intrinsic biochemical properties and sites of autophosphorylation of a plant protein kinase.