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. 1990 Jun;172(6):3257–3263. doi: 10.1128/jb.172.6.3257-3263.1990

Overproduction, purification, and characterization of Bacillus subtilis RNA polymerase sigma A factor.

B Y Chang 1, R H Doi 1
PMCID: PMC209133  PMID: 2111806

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.

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