Vrentas, CE, Gaal, T, Burgess, RR and Gourse, RL (2010) An improved procedure for the purification of the Escherichia coli RNA polymerase omega subunit. Protein Expr. Purif. 71:190-4
We report an improved procedure for purification of the omega subunit of Escherichia coli RNA polymerase. In contrast to the original procedure, the revised procedure: (i) allows purification of omega entirely from the soluble fraction, obviating the need for denaturation/renaturation, (ii) results in >99% pure omega in only two chromatographic steps, and (iii) improves the yield of purified omega by at least 5-fold. Reconstitution of E. coli RNAP from omega purified by this procedure, as well as purified sigma and core RNAP lacking omega, produces active holoenzyme in vitro, and co-overexpression of omega from a plasmid containing rpoZ and an additional plasmid encoding the other RNAP core subunits results in production of active core enzyme in vivo.
DNA-Directed RNA Polymerases/genetics; DNA-Directed RNA Polymerases/isolation & purification; DNA-Directed RNA Polymerases/metabolism; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli/isolation & purification; Plasmids/genetics; Plasmids/metabolism
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