title: Overproduction, purification, and characterization of Bacillus subtilis RNA polymerase sigma A factor.
abstract: By use of a T7 expression system, large amounts of active Bacillus subtilis RNA polymerase sigma A factor were produced in Escherichia coli cells. This overproduced protein was found in the form of inclusion bodies and constituted 40% of the total cellular protein. Because of the ease of isolation of the inclusion bodies and the acidic properties of sigma A, the protein was purified to more than 99% purity and the yield was about 90 mg/liter of culture. Gel mobility, antigenicity, specificity of promoter recognition, and N-terminal amino acid sequence of the overproduced sigma were found to be the same as those of native sigma A. Partial proteolysis analysis of sigma A protein suggested the presence of a protease-sensitive surface region in the C-terminal part of the sigma A protein. The promoter -10 binding region of sigma A was less sensitive to proteases and was probably involved in a hydrophobic, tightly folded domain of sigma A protein.
authors.affiliation: Department of Biochemistry and Biophysics, University of California, Davis 95616.
authors.forename: B Y
authors.initials: BY
authors.lastname: Chang
authors.forename: R H
authors.initials: RH
authors.lastname: Doi
mesh_terms: D001412:Bacillus subtilis / Q000032:analysis* / Q000235:genetics; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis*; D008970:Molecular Weight; D010449:Peptide Mapping; D012808:Sigma Factor / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D014157:Transcription Factors / Q000302:isolation & purification*; D014158:Transcription, Genetic
chemical_list: D012808:Sigma Factor; D014157:Transcription Factors; D012321:DNA-Directed RNA Polymerases