INTRODUCTIONA wide variety of plasmid vectors are commercially available for the production of fusion proteins in bacterial cells. Most are also designed to incorporate a tag that allows affinity purification of the expressed fusion protein from bacterial cell extracts. The most commonly used are vectors that incorporate a portion of the glutathione-S-transferase (GST) enzyme that is able to bind to immobilized glutathione and vectors that use a polyhistidine tag which binds immobilized nickel ions with high affinity. This protocol describes preparation of an insoluble GST fusion protein, isolated using glutathione agarose beads.