OVERPRODUCTION, PURIFICATION, AND CHARACTERIZATION OF BACILLUS-SUBTILIS RNA-POLYMERASE SIGMA-A FACTOR

By use of a T7 expression system, large amounts of active Bacillus subtilis RNA polymerase σ(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 σ(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 σ were found to be the same as those of native σ(A). Partial proteolysis analysis of σ(A) protein suggested the presence of a protease-sensitive surface region in the C-terminal part of the σ(A) protein. The promoter -10 binding region of σ(A) was less sensitive to proteases and was probably involved in a hydrophobic, tightly folded domain of σ(A) protein.