结核分枝杆菌甲硫氨酰tRNA合成酶与底物及其类似物的结合比较

Comparison of the binding of substrates and analogs to Mycobacterium tuberculosis MetRS

目的建立重组结核分枝杆菌甲硫氨酰tRNA合成酶(MetRS)的制备方法,分析底物及其类似物与重组蛋白的结合能力。方法将结核分枝杆菌MetRS(MtMetRS)基因置于不同启动子调控之下。通过表达筛选选择重组蛋白大量制备的条件,使用分子筛制备重组蛋白,并验证其分子量。使用Thermal Shift Assay比较MetRS底物及其类似物与重组结核分枝杆菌MetRS的结合能力。结果通过不同重组质粒的表达筛选,确定重组蛋白制备方法。Thermal Shift Assay分析提示甲硫基腺苷酸(methionyl-adenylate,MetAde)与焦磷酸同时存在时可以提高重组蛋白的热稳定性。结论成功制备出具有天然活性的重组MtMetRS蛋白;应以甲硫基腺苷酸和焦磷酸分子结构为方向,设计、筛选结核分枝杆菌MetRS抑制剂。

The MetRS gene of Mycobacterium tuberculosis is under the control of different promoters.The conditions for mass production of recombinant proteins were selected through expression screening in Escherichia coli,and gel filtration chromatography was used to prepare recombinant proteins.Thermal shift assays were used to evaluate the binding ability of MetRS substrate and its analogues to recombinant Mycobacterium tuberculosis MetRS.These assays showed that the co-existence of methionyl-adenylate and pyrophosphate improved the thermal stability of the recombinant protein.