Overproduction and Purification of σ 32, the Escherichia coli Heat Shock Transcription Factor

Abstract

This paper reports the overproduction and the details of a rapid method to purify active σ 32 that is free of core RNA polymerase enzyme. Maximal overproduction of σ 32 in a T7 RNA polymerase-based expression system is achieved only in the presence of rifampicin. This 2-day procedure involves solubilizing inclusion bodies in Sarkosyl, removal of Sarkosyl hy dialysis, and a single S-Sepharose column chromatography step. The final yield of σ 32 is about 4.1 mg of approximately 95% purity from 1 g of wet weight cells.