Separation of copurifying GroEL from glutathione-S-transferase fusion proteins

The purification of overexpressed fusion proteins using bacterial expressio n systems is a useful tool for the study of many proteins. One problem that can occur is the formation of stable interactions between the expressed fu sion protein and certain endogenous bacterial proteins, such as the molecul ar chaperone GroEL. Such interactions may result in the copurification of c ontaminating bacterial proteins. Here we describe an efficient and inexpens ive method for the removal of contaminating GroEL from a bacterially expres sed GST fusion protein, in this method, denatured bacterial proteins are ad ded to the bacterial lysates prior to the addition of glutathione Sepharose resin. The denatured proteins compete for GroEL binding, thereby releasing the GroEL contaminants from the expressed fusion protein. (C) 2000 Academi c Press.