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

AUTOR(ES)

Chang, B Y

RESUMO

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.

Documentos Relacionados

• Structure of a Bacillus subtilis bacteriophage SPO1 gene encoding RNA polymerase sigma factor.
• Isolation and characterization of the Bacillus subtilis sigma 28 factor.
• Overproduction, purification, and biochemical characterization of a xylanase (Xys1) from Streptomyces halstedii JM8.
• Novel RNA polymerase sigma factor from Bacillus subtilis.
• Purification, Overproduction, and Partial Characterization of β-RFAP Synthase, a Key Enzyme in the Methanopterin Biosynthesis Pathway†