为了获得可用于X射线衍射的恶臭假单胞菌尼古丁代谢途径中关键单加氧酶HspB的单晶。定点突变PCR构建重组质粒，大肠杆菌中诱导表达，镍柱亲和层析、烟草蚀纹病毒（Tobacco etch virus，TEV）蛋白酶酶切和凝胶过滤层析纯化，悬滴扩散法进行结晶。成功构建重组质粒并获得高表达；比较了TEV蛋白酶柱上及透析酶切的效率，TEV蛋白酶透析酶切效率更高；确定了该纯化路线，获得电泳纯级的HspB蛋白。结晶条件初筛和正交优化后获得可培养HspB蛋白单晶的条件为22% PEG3350、0.1 mol/L Bis-Tris pH6.5、0.21 mol/L MgCl2、18℃、150比例加晶种。去除标签后的HspB蛋白获得了分辨率1.8Å的单晶。%It was to obtain the monocrystal of HspB, the key monooxygenase in the nicotine degradation pathway fromPseudomonas putida, for X-ray diffraction. The expression plasmid was constructed by site-directed mutagenesis PCR and was expressed in Escherichia coli. Target protein was purified with immobilized metal-chelating affinity chromatography, TEV protease digestion and gel filtration chromatography. Crystal culture though hanging-drop vapor-diffusion method. The recombinant plasmid was successfully constructed and expressed high. Compared to digestion on the column, digestion in the solution during dialysis is more efficient, and the purification strategy was determined to obtain HspB with high purity. The optimal crystallization condition was identified as 22% PEG3350, 0.1 mol/L Bis-Tris pH6.5, 0.21 mol/L MgCl2, 18℃, seeding after orthogonal experiments. HspB with His-tag cleavaged can obtain a monocrystal which resolution reaches 1.8Å.
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