A system for heterologous expression and isolation of Escherichia coli RNA polymerase and its components

Abstract

A set of plasmid vectors for expression of all major Escherichia coli RNA polymerase subunits as fusion proteins with intein- and chitin-binding domains, allowing protein purification in accordance with IMPACT technology, was constructed. It is demonstrated that the fusion subunits alpha, beta or beta' in conjunction with the natural subunits alpha, beta, beta', and sigma can participate in RNA polymerase assembly in vivo, providing affinity-based isolation of the enzyme. Functional activity of the enzyme preparations was demonstrated in the experiments on in vitro transcription and promoter complex formation. With the use of IMPACT technology, sigma(70) subunit can be isolated as an individual protein without admixture of RNA polymerase.

Purification of Escherichia coli RNA polymerase using a self‐cleaving elastin‐like polypeptide tag

“…Furthermore, it was confirmed that this method is compatible with PEG precipitation to remove most nonspecifically bound polynucleotides. Thus this method provides a simple, economical alternative to previously reported single‐step RNAP purification methods involving immunoaffinity chromatography11 or the intein‐based IMPACT‐CN chromatography system 12, 13…”

“…Among these is the potential for dissociation of the target complexes during elution from the affinity matrix, and unexpected nonspecific cleavage within the target protein by protease during protease‐mediated affinity tag removal. Although new methods based on gentle immunoaffinity,11 and self‐cleaving chitin‐binding tags have been successfully used to purify protein complexes, including Escherichia coli RNA polymerase (RNAP),12, 13 they still rely on expensive chromatographic methods.…”