结核分枝杆菌甲硫氨酰tRNA合成酶的结晶方法

Crystallization method of Mycobacterium tuberculosis methionyl tRNA synthetase

目的 制备结核分枝杆菌甲硫氨酰tRNA合成酶(Mt MetRS)晶体。方法 设计不同的重组Mt MetRS蛋白,使用大肠杆菌表达重组蛋白。坐滴气相扩散法在293 K温度下筛选不同重组蛋白的结晶条件,并使用悬滴法进行晶体优化。优化的晶体经液氮速冻保存。在上海同步辐射光源进行晶体衍射数据采集。结果 使用不同设计的重组蛋白进行结晶筛选和优化,最终获得了可以进行晶体衍射的高分辨率蛋白质晶体。并确定6×His序列在Mt MetRS分子的结晶中起到关键作用。结论 Mt MetRS分子两端的6×His序列是帮助Mt MetRS分子结晶的重要因素。

Objective To construct the crystal of Mycobacterium tuberculosis methionyl tRNA synthetase(Mt MetRS). Methods Different recombinant Mt MetRS proteins were designed and expressed by Escherichia coli.Crystallization conditions were initially searched using the sitting-drop vapour-diffusion method at 293 K, and optimization was carried out by the hanging-drop vapour-diffusion method. The optimized crystals were preserved by using liquid nitrogen freezing. The diffraction data were collected at the Shanghai Synchrotron Radiation Light Source. Results X-ray quality crystals were obtained by crystallization screening and optimization with different designs of recombinant proteins. The 6×His sequence played a key role in the crystallization of Mt MetRS molecules. Conclusion The 6×His sequence at both ends of Mt MetRS molecule is an important factor to facilitate crystallization.