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.
Aniskovitch
Lyudmila P
LP
Laboratory of Molecular Biology, Department of Microbiology and Immunology, University of South Alabama College of Medicine, Mobile, AL 36688, USA.
Winkler
Herbert H
HH
Laboratory of Molecular Biology, Department of Microbiology and Immunology, University of South Alabama College of Medicine, Mobile, AL 36688, USA.
D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012283:Rickettsia prowazekii / Q000235:genetics* / Q000378:metabolism*; D012808:Sigma Factor / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D012808:Sigma Factor; D012321:DNA-Directed RNA Polymerases