结核分枝杆菌耐药性相关膜蛋白MmpL6的表达与纯化

Expression and purification of multidrug resistance related membrane protein MmpL6 of Mycobacterium tuberculosis

目的构建结核分枝杆菌外排泵蛋白MmpL6(Rv1557)表达载体,在大肠杆菌E.coli中诱导MmpL6蛋白高表达并进行纯化。方法以结核分枝杆菌H37Rv菌株基因组DNA为模板,采用聚合酶链反应扩增目的基因片段,构建pET21b-MmpL6表达载体。将表达载体转化至大肠杆菌中,加入异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导重组蛋白表达,并针对不同表达菌株、温度进行优化,获取最优表达条件。采用Ni-NTA亲和层析以及凝胶过滤色谱纯化目标蛋白。结果成功构建pET21b-MmpL6重组表达质粒,经IPTG诱导后,在Rosetta菌株中25℃条件下获得最高表达量。结论成功表达并纯化了MmpL6蛋白,为该蛋白后续的结构与功能研究奠定了基础。

Objective To construct the prokaryotic expression vector carrying Mycobacterium tuberculosis gene mmpL6 （rv1557）, overexpress and purify the recombinant MmpL6 （Rv1557） protein in Escherichia coli （E. coli）. Methods The DNA fragment of MmpL6 was amplified by polymerase chain reaction using the genomic DNA of Mycobacterium tuberculosis H37Rv strain as a template. The target gene was cloned into pET21b vector to construct the pET21b-MmpL6 expression plasmid, and then transformed into E. coil for protein expression. Recombinant protein expression was induced by isopropyl β-D-thiogalactopyranoside （IFFG） and detected by SDS-PAGE combined with Western blot. The overexpressed MmpL6 protein was purified by Ni-affinity chromatography and gel filtration chromatography. Results The recombinant pET21 b-MmpL6 plasmid was successfully constructed and the highest expression level was obtained at 25 ℃ in Rosetta strain induced by IFFG. Conclusion The successful expression and purification of MmpL6 in E. coli lays the foundation for the further structure and function studies of the protein, and also provides clues to the design of anti-tuberculosis drugs targeting efflux pump proteins.