Overexpression and purification of a biologically active rifampicin-resistant beta subunit of Escherichia coli RNA polymerase.
The gene rpoB (rifD 18), which encodes rifampicin-resistant beta subunit of Escherichia coli RNA polymerase, has been placed on an overexpression plasmid under the control of bacteriophage T7 promoter. Induction of the T7 RNA polymerase gene in the host cells resulted in extensive overproduction of the beta polypeptide. Most of the overproduced material was recovered from cell lysates in insoluble form and was solubilized by extraction with 6 M urea. Purified overproduced beta subunit was added, in molar excess, to urea-denatured rifampicin-sensitive RNA polymerase. Upon removal of urea by dialysis, the reconstituted enzyme became rifampicin-resistant, indicating that overproduced beta subunit can be efficiently assembled into functional holoenzyme.