Taguchi S, Kumagai I, Miura K
Department of Industrial Chemistry, Faculty of Engineering, University of Tokyo, Japan.
Biochim Biophys Acta. 1990 Jul 30;1049(3):278-85. doi: 10.1016/0167-4781(90)90098-m.
To elucidate differences in the mechanism of gene expression between Streptomyces and Escherichia coli, the regulatory region for expression of the gene for a proteinaceous proteinase inhibitor, Streptomyces subtilisin inhibitor (SSI), was altered to express efficiently in E. coli. This was carried out by inserting a pre-SSI-encoding region downstream of the tac promoter and ribosome-binding site in a multi-copy plasmid. When the resultant plasmid pMKSI161-9 was introduced into E. coli JM105, SSI protein was found to be expressed and secreted into the periplasmic space by Western blot analysis. When introduced into 'leaky' E. coli strains, this protein was detected in the medium as well as in the periplasmic space in bacteria. NH2-terminal sequencing analysis of the SSI purified from E. coli JM105 indicated two processing sites, Ala(-4)/Ala(-3)-Pro(-2)-Gly and Ala(-4)-Ala-3/Pro(-2)-Gly-1, of pre-SII. These sites were different from those in Streptomyces albogriseolus S-3253 and Streptomyces lividans 66. The inhibitor activity of the processed protein toward subtilisin BPN' was almost the same as that of authentic SSI.
为阐明链霉菌和大肠杆菌基因表达机制的差异，对一种蛋白质类蛋白酶抑制剂——链霉菌枯草杆菌蛋白酶抑制剂（SSI）基因的表达调控区进行改造，使其能在大肠杆菌中高效表达。具体做法是将编码前体SSI的区域插入多拷贝质粒中tac启动子和核糖体结合位点的下游。将所得质粒pMKSI161 - 9导入大肠杆菌JM105后，通过蛋白质印迹分析发现SSI蛋白得以表达并分泌到周质空间。将该质粒导入“渗漏型”大肠杆菌菌株后，在培养基以及细菌的周质空间中均检测到了这种蛋白。对从大肠杆菌JM105中纯化得到的SSI进行氨基末端测序分析，结果表明前体SII存在两个加工位点，即Ala(-4)/Ala(-3)-Pro(-2)-Gly和Ala(-4)-Ala-3/Pro(-2)-Gly-1。这些位点与浅灰链霉菌S - 3253和变铅青链霉菌66中的位点不同。加工后的蛋白对枯草杆菌蛋白酶BPN'的抑制活性与天然SSI几乎相同。
