Recombinant Escherichia coli RNA polymerase: purification of individually overexpressed subunits and in vitro assembly

New improved methods were developed for the purification to apparent homogeneity of alpha, beta, beta', and sigma subunits of Escherichia coli RNA polymerase (RNAP) from corresponding overproducing strains. The purified subunits were assembled into enzymatically active RNAP holoenzyme (alpha 2 beta beta' sigma) using the optimal subunit molar ratio (alpha:beta:beta':sigma = 2:8:4:1) at a total protein concentration of 0.5 mg/ml. The presence of sigma subunit and 10 microM ZnCl2 in the reconstitution mixture increased the yield of RNAP approximately 4 times. The assembled RNA polymerase was purified by two successive chromatographic steps using size-exclusion Superose 6 and anion exchange Mono Q FPLC columns, which resulted in the electrophoretically homogeneous holoenzyme with overall yield of 56%. The specific activity of the recombinant RNAP estimated by the standard T4 transcription assay was 6.5 nmol of [3H]UTP incorporated into acid-insoluble RNA product per microgram of RNAP per 1 h.