Rickettsia prowazekii sigma factor sigma(73) can be overexpressed in Escherichia coli and promotes RNA polymerase binding and transcription

The sigma factor sigma(73) of the obligate intracytoplasmic bacterium Rickettsia prowazekii was overexpressed and purified from Escherichia coli. The rickettsial rpoD gene encoding sigma(73) was cloned into a Ndel-BamHI-cleaved pET-15b vector under control of T7 transcription and translation signals. The recombinant plasmid encoded a 75 kDa fusion protein that was overproduced in E. coli BL21(DE3) and purified from inclusion bodies after solubilization with guanidine hydrochloride and using His.Bind metal chelation resin. The N-terminal His.Tag sequence of the 75 kDa fusion protein was removed by thrombin treatment to obtain R. prowazekii sigma(73T). The R. prowazekii sigma(73T) as well as the 75 kDa fusion protein had the ability to bind to core DNA-dependent RNA polymerase of both R. prowazekii and E. coli and to stimulate their interaction with a rickettsial promoter.