Overproduction and purification of the ω subunit of Escherichia coli RNA polymerase

Abstract

This paper reports the construction of plasmids which direct the overproduction of the w subunit of Escherichia coli RNA polymerase and the subsequent purification of ω. Useful overproduction is achieved only if the natural ribosomal binding site region of rpoZ is replaced with the ribosomal binding site region of bacteriophage T7 gene 10. Overproduction is directed by T7 RNA polymerase which is provided on a separate plasmid. ω is purified by three column steps either from the insoluble inclusion body fraction or from the soluble fractions of lysates. The final yield is approximately 2 mg ω per 10 g cells wet wt. Additionally, we found that recombinant ω is readily cleaved by an endogenous protease. Sequence analysis of the most prevalent proteolytic fragment suggested that the protease responsible was the product of the ompT gene. Cleavage of ω is greatly reduced in ompT − strains.