FIG. 1.

Preparation of recombinant RNAP from Anabaena sp. strain PCC 7120. Preparations of His-tagged α and σ (SigA) subunits of Anabaena RNAP expressed in E. coli and purified (A) and of solubilized inclusion bodies from E. coli containing the β, β′, and γ subunits (B), all of them obtained under denaturing conditions, were subjected to SDS-PAGE and stained with Coomassie brilliant blue R. Portions of the preparations of His-tagged α, β, β′, and γ subunits were mixed and renatured together. The resulting preparation was mixed with a preparation of renatured His-SigA, and the obtained reconstituted RNAP holoenzyme was purified by chromatography through chelating Sepharose. Preparations obtained after renaturation (C) and holoenzyme purification (D) were also analyzed by SDS-PAGE. See Materials and Methods for further details. Size standards (SS) in kDa are shown.