12] Purification of overproduced Escherichia coli RNA polymerase σ factors by solubilizing inclusion bodies and refolding from Sarkosyl

Abstract

This chapter summarizes a method for solubilizing and refolding proteins from inclusion bodies utilizing the mild anionic detergent, Sarkosyl. This method is found to be helpful in refolding several polymerase subunits and sigma (σ) factors with recoveries of monomeric refolded protein of greater than 50%. To obtain purified, active, monomeric protein, one must isolate the inclusion bodies, solubilize the protein in the inclusion bodies, and then allow the solubilized protein to refold by gradually removing the solubilizing agent. This approach has been used to isolate overproduced components of the E. coli transcription machinery. Most of the early work involved solubilizing with denaturants such as 8 M urea or 6 M guanidinium hydrochloride and refolding by slow dilution. It was commonly found that, on dilution to low denaturant, most of the protein aggregated and precipitated. Even when no visible precipitate formed, there was often soluble multimer present in the diluted solution. This was subsequently lost when the apparently soluble protein was bound to an ionexchange column and subjected to salt gradient elution. The majority of the refolded protein was multimeric, bound tightly to the column, and was never recovered.