Steffensen S, Poulsen A B, Mortensen K K, Korsager B, Sperling-Petersen H U
Department of Chemistry, Aarhus University, Denmark.
Biochem Mol Biol Int. 1994 Dec;34(6):1245-51.
During purification of the translation initiation factor IF2 from ompT+ strains of Escherichia coli the IF2 is partially degraded in the presence of membrane debris during the first steps of purification. This is a result of proteolytic cleavage by outer membrane protease OmpT [1]. Here we have investigated the activity of OmpT in 51 clinical E. coli isolates of human origin, by a time dependent OmpT activity assay using IF2 as target protein. The activity of OmpT in the outer cell membrane is highly variable among wild type E.coli strains, ranging from no detectable activity in 65% of the strains to a very high activity in 5 strains (10%). The OmpT activity is closely related to the assay temperature and to the growth temperature of the cells, and can be reduced or even eliminated by lowering the temperature of growth. The results open the possibility of using non-denaturing gel electrophoresis of crude cell lysates as a screening method in population genetic studies of initiation factor IF2 and other cytoplasmic proteins which are cleaved by OmpT.
在从大肠杆菌ompT⁺菌株中纯化翻译起始因子IF2的过程中，IF2在纯化的第一步，即在存在膜碎片的情况下会部分降解。这是外膜蛋白酶OmpT进行蛋白水解切割的结果[1]。在此，我们通过以IF2作为靶蛋白的时间依赖性OmpT活性测定法，研究了51株源自人类的临床大肠杆菌分离株中OmpT的活性。在外细胞膜中，OmpT的活性在野生型大肠杆菌菌株中差异很大，65%的菌株检测不到活性，而5株菌株（10%）活性非常高。OmpT活性与测定温度以及细胞的生长温度密切相关，通过降低生长温度可以降低甚至消除该活性。这些结果为在起始因子IF2和其他被OmpT切割的细胞质蛋白的群体遗传学研究中，将粗细胞裂解物的非变性凝胶电泳用作筛选方法提供了可能性。
