大肠杆菌离体重组系统转运FhaB突变体

Escherichia coli in vitro recombinant system transports FhaB mutants

目的探讨离体重组系统用于双组分转运系统机制研究的可行性。方法通过构建不同分子质量大小的FhaB突变体,制备成原生质球,加入到FhaC蛋白脂质体中,使用蛋白酶K消化的方法检测所构建的离体重组体系转运不同大小底物的能力。结果成功构建突变体后,检测到原生质球分泌的37、60和80 ku大小的条带,并且含FhaC的蛋白脂质体能转运这3种分子质量大小的底物,此次所转运的底物分子质量高达80 ku。结论体外构建的重组转运系统可以转运大分子质量的底物,可尝试使用该体系进行双组分系统机制的研究。

Objective To explore the mechanism of two-partner secretion with an in vitro reconstitution system.Methods Spheroplasts were used to construct FhaB mutants with different molecular weights,and then combined with FhaC containing proteoliposomes to perform the translocation experiments followed by digestion with proteinase K to test the transportation of substrates with different sizes in vitro by this recombinant system.Results After successful construction of the mutant,37 ku,60 ku and 80 ku bands secreted by spheroplasts were detected,and FhaC-containing protein liposomes could transport these three substrates with molecular weight as high as 80 ku.Conclusions The recombinant transport system constructed in vitro may transport substrates with large molecular weight and the mechanism of the two-partner secretion can be studied by this system.