Overproduction and purification of recombinant Bacillus subtilis RNA polymerase

We describe a vector-based system for the production of recombinant Bacillus subtilis RNA polymerase. The recombinant enzyme is C-terminally tagged with nine consecutive histidine residues resulting in about 90% pure enzyme in a single nickel-affinity purification step. The vectors permitted production of recombinant enzyme lacking an ω subunit or containing either the ω1 (YkzG) or ω2 (YloH) subunits. In transcription time-course assays all of the recombinant enzymes exhibited identical activity to native RNAP. The modular assembly of the artificial RNA polymerase operon permits ready mutation of any subunit and incorporation into the recombinant enzyme, which will enable new functional/structural studies with this enzyme.