Purification of Highly-Active and Soluble Escherichia coli σ70 Polypeptide Overproduced at Low Temperature

Abstract

Publisher Summary This chapter explores the sigma subunit of bacterial RNA polymerase (RNAP) that is required for the promoter-specific transcription initiation. It is a very common phenomenon, that a high-level expression of desired proteins is accompanied by a deposition of overproduced products in insoluble inclusion bodies in E. Coli-based expression systems. The reasons for misfolding and subsequent aggregation of overproduced proteins in E. coli are not well understood so far; however, it has been reported that the solubility and activity of overproduced proteins can be increased by induction at low temperature, fusion with thioredoxin, or coexpression with some molecular chaperones. The chapter aims to describe a simple method for the purification of large amounts of soluble σ70 polypeptide with high activity. The key element to obtain an overproduced soluble σ70 polypeptide inside cells is to induce the expression of the protein at a low temperature instead of at 37o. Because of this method, isolating highly pure and active Histag- σ70 recombinant proteins in larger quantities using only two simple steps is possible. A schematic of the overproduction and preparation procedure is also explained.