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  • Title: Rickettsia prowazekii sigma factor sigma 73 can be overexpressed in Escherichia coli and promotes RNA polymerase binding and transcription.
    Author: Aniskovitch LP, Winkler HH.
    Journal: Microbiology (Reading); 1996 Apr; 142 ( Pt 4)():901-906. PubMed ID: 8936316.
    Abstract:
    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.
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