Kumamoto C A, Chen L, Fandl J, Tai P C
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
J Biol Chem. 1989 Feb 5;264(4):2242-9.
Mutations in the Escherichia coli secB gene lead to protein export defects in vivo (Kumamoto, C.A., and Beckwith, J. (1985) J. Bacteriol. 163, 267-274). To demonstrate directly the participation of the secB gene product (SecB) in protein export, SecB was purified, and its effects on in vitro protein translocation were analyzed. SecB was purified from soluble extracts of a strain that overproduced it, by ammonium sulfate precipitation, DEAE-cellulose chromatography, and differential precipitation at acid pH. The chromatographic behavior on gel filtration columns indicated apparent molecular masses of approximately 90 kDa for both purified SecB and SecB in cytosolic extracts of wild type cells. When added to a translocation mixture, purified SecB stimulated pro-OmpA translocation into membrane vesicles. SecB also suppressed the thermoinduced defect in translocating activity of membranes derived from a secY24 mutant. The results of these in vitro studies and of previous in vivo studies demonstrate that SecB plays a direct role in normal protein export in E. coli.
大肠杆菌secB基因的突变会导致体内蛋白质输出缺陷（熊本，C.A.，和贝克威思，J.（1985年）《细菌学杂志》163，267 - 274）。为了直接证明secB基因产物（SecB）参与蛋白质输出，对SecB进行了纯化，并分析了其对体外蛋白质转运的影响。通过硫酸铵沉淀、DEAE - 纤维素色谱法以及酸性pH下的差异沉淀，从过量表达SecB的菌株的可溶性提取物中纯化出SecB。凝胶过滤柱上的色谱行为表明，纯化的SecB和野生型细胞胞质提取物中的SecB的表观分子量约为90 kDa。当添加到转运混合物中时，纯化的SecB刺激前OmpA转运到膜泡中。SecB还抑制了源自secY24突变体的膜在转运活性方面的热诱导缺陷。这些体外研究结果和先前的体内研究结果表明，SecB在大肠杆菌正常蛋白质输出中起直接作用。
