大肠杆菌外膜蛋白酶T及其突变体的表达、复性及生物活性分析

Expression, refolding and characterization of Escherichia coli outer membrane T and its mutant

摘要:

外膜蛋白酶T (Outer-membrane protease T, OmpT)是定位于大肠杆菌外膜, 具有高度底物特异性的蛋白水解酶。本文旨在建立克隆表达膜蛋白OmpT和体外复性的方法, 考察其蛋白酶活性。首先以大肠杆菌基因组DNA为模板, PCR扩增ompT基因, 连接至pET28a (pET-ompT), 引入点突变Asp85Ala, 构建表达质粒pET-ompT85。然后将两种重组质粒转化入BL21 (DE3), 均以包涵体形式大量表达。纯化后的蛋白经稀释法复性, 并加入粗制脂多糖(Lipopolysaccharide, LPS)恢复蛋白酶活性。通过SDS-PAGE、鱼精蛋白水解试验及生长曲线观察表明, 重组蛋白OmpT在体外能水解抗菌肽鱼精蛋白和兔肌肉肌酸激酶, 而OmpT突变体则无上述功能。上述结果表明本文获得了具有蛋白水解酶功能的重组蛋白OmpT, 该蛋白在体外可保护大肠杆菌抵抗鱼精蛋白的杀菌作用。

Abstract:

OmpT, located in Escherichia coli (E. coli) outer membrane, is a protease that demonstrates highly substrate specificity. In order to estalish the approaches for expression and refolding of membrane protein OmpT, and examine the demonstrated protease activity of OmpT, the ompT gene was first amplified by PCR and inserted into pET28a (pET-ompT) and introduced by Asp85Ala site-directed mutagenesis to generate mutant Asp85Ala (pET-ompT85). Then, the two recombinant plasmids were transformed into BL21 (DE3), OmpT and the mutant were expressed in the form of inclusion bodies, purified and refolded by N-Dodecyl-N,N-dimethyl-1-ammonio-3-propanesulphonate. The addition of lipopolysaccharide (LPS) to the recombinant OmpT was critical to refold its protease activity in vitro. Finally, the ability of the recombinant wide-type OmpT to hydrolyze protamine and rabbit muscle creatine kinase (RMCK) was confirmed by SDS-PAGE, bacteria agglutination and growth curve in contrast to the mutant. The results suggest that the desired recombinant OmpT was obtained, which showed the significant protease activity in the protection of E. coli against protamine in vitro.