Chang B Y, Doi R H
Department of Biochemistry and Biophysics, University of California, Davis 95616.
J Bacteriol. 1990 Jun;172(6):3257-63. doi: 10.1128/jb.172.6.3257-3263.1990.
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.
通过使用T7表达系统，在大肠杆菌细胞中产生了大量活性枯草芽孢杆菌RNA聚合酶σA因子。这种过量产生的蛋白质以包涵体的形式存在，占细胞总蛋白的40%。由于包涵体易于分离且σA具有酸性特性，该蛋白质被纯化至纯度超过99%，产量约为90毫克/升培养物。发现过量产生的σ的凝胶迁移率、抗原性、启动子识别特异性和N端氨基酸序列与天然σA相同。σA蛋白的部分蛋白酶解分析表明，在σA蛋白的C端部分存在一个对蛋白酶敏感的表面区域。σA的启动子 -10结合区域对蛋白酶不太敏感，可能参与了σA蛋白的一个疏水、紧密折叠的结构域。
