Expression and purification of GST fusion proteins

Abstract

This unit describes the use of the glutathione-S-transferase (GST) gene fusion system as a method for high-level protein expression and purification from bacterial lysates. Several pGEX vectors are available with multiple cloning sites to allow for unidirectional insertion of the coding-region DNA into the pGEX vector. The GST fusion protein is easily purified by affinity chromatography using a glutathione-Sepharose matrix under mild conditions. Removal of the GST moiety from the protein of interest is accomplished through a specific protease cleavage site located between the GST moiety and the recombinant polypeptide. For solution digestions, GST is easily removed by a second round of chromatography on the glutathione column. Removal of proteases is facilitated by the use of a benzamidine-Sepharose column or a gel-filtration step. Purified protein has been used successfully in structural determinations, immunological studies, vaccine production, and structure-function analysis of protein-protein or DNA-protein interactions.