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目的优化结核分枝杆菌wbbL基因在大肠埃希菌中的表达,获得大量可溶性产物并加以鉴定。方法不同条件下诱导pET16b-wbbL质粒在大肠埃希菌BL21(DE3)中的表达;建立外源重组蛋白质与分子伴侣在大肠埃希菌中的共表达体系,优化其表达条件;超声破碎诱导表达的大肠埃希菌,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表达产物;免疫蛋白印迹(Western blotting)法对重组蛋白质进行鉴定。结果共表达体系优化表达的蛋白质主要以可溶性形式存在,经0.5 g/L L-阿拉伯糖30℃诱导5 h、0.5 mmol/L异丙酸-D-硫代半乳糖苷30℃诱导2 h时,可溶性目的蛋白的产量最多,占细胞蛋白总量的55.6%,且所表达的蛋白质为wbbL基因产物。结论在大肠埃希菌中成功表达出大量可溶性结核分枝杆菌wbbL蛋白质,为研究其酶促反应动力学特征以及建立完善的筛选抗结核药物的酶反应体系提供了基础。
Objective To optimize the expression of wbbL gene of M. tuberculosis in Escherichia coli and obtain a large number of soluble products and identify them. Methods The expression of pET16b-wbbL plasmid was induced in Escherichia coli BL21 (DE3) under different conditions. The co-expression system of exogenous recombinant protein and molecular chaperone in Escherichia coli was established and its expression conditions were optimized. The expressed product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The recombinant protein was identified by Western blotting. Results The protein expressed by co-expression system mainly existed in soluble form. After induced by 0.5 g / L L-arabinose at 30 ℃ for 5 h and 0.5 mmol / L isopropionic acid-D-thiogalactopyranoside at 30 ℃ for 2 h , The soluble protein of interest is the most abundant, accounting for 55.6% of the total cellular proteins, and the expressed protein is the wbbL gene product. Conclusion A large number of soluble Mycobacterium tuberculosis wbbL protein was successfully expressed in Escherichia coli, which provided the basis for studying its enzymatic kinetic characteristics and establishing a perfect enzyme reaction system for screening anti-TB drugs.