Expression and purification of glutathione-S-transferase fusion proteins

This unit describes how pGEX vectors can be used in bacterial systems to express foreign polypeptides as fusions with glutathione-S-transferase (GST). In general, such fusion proteins are soluble and are easily purified from lysed cells under nondenaturing conditions by absorption with glutathione-agarose beads, followed by elution in the presence of free glutathione. Potential applications of the pGEX vectors include the expression and purification of individual polypeptides (including short peptides) for use as immunogens and as biochemical and biological reagents, and in the construction of cDNA expression libraries. This protocol describes production and screening of pGEX transformants and purification of milligram quantities of fusion proteins from 1-liter cultures. The commentary describes several modifications to the expression and purification protocol that may be useful in cases where fusion proteins are insoluble or unstable.