12] Micro- and macropurification methods for protein kinases

Abstract

Publisher Summary This chapter describes various methods used to identify and purify a rare protein kinase, assuming its mode of activation is known, and the problems that may be encountered in scaling up from micro- to macropurification. The first step in working with a protein kinase is to establish a specific and rapid assay for the enzyme. The most important component in the assay is the protein substrate, which can be an endogenous cellular target protein, a synthetic peptide based on the sequence phosphorylated in this protein, or a model substrate protein. Following the phosphorylation reaction, one of the three widely used methods can be employed to separate the substrate from nucleotide triphosphates: trichloroacetic acid (TCA) precipitation, spotting of positively charged substrates on phosphocellulose paper, and detection of phosphorylated substrates by gel electrophoresis. To purify a protein kinase from a limited source, the most useful strategy is to remove abundant proteins by classical chromatographic techniques before applying affinity steps; remove inhibitors or inactivators at an early stage in the purification; keep sample volumes to a minimum while avoiding dialysis; and move through the purification rapidly without freezing the enzyme. The classical chromatographic techniques used to remove bulk protein include cation and anion-exchange, hydrophobic, and gel-filtration chromatography. At the end of a micropurification, the purity of the protein kinase should be checked on a silver-stained sodium dodecyl sulfate (SDS)-polyacrylamide gel. While scaling up the purification of a protein kinase the first problems encountered are the heterogeneity of proteins present in tissue extracts, the higher amount of starting material, and the large volumes of crude extract. One can use ammonium sulfate precipitation to reduce volumes and gain some purification.