恶臭假单胞菌HspB的单晶培养及结晶条件优化  

Monocrystal Culture and Crystallization Conditions Optimization of Hsp B from Pseudomonas putida

作　　者：邬志杰 吴更[1] 唐鸿志[1] 许平[1] 

机构地区：[1]上海交通大学生命科学技术学院微生物代谢国家重点实验室,上海200240

出　　处：《生物技术通报》2015年第11期236-242,共7页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31230002;31470223)

摘　　要：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Hsp B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Hsp B蛋白。结晶条件初筛和正交优化后获得可培养Hsp B蛋白单晶的条件为22%PEG3350、0.1 mol/L Bis-Tris p H6.5、0.21 mol/L Mg Cl2、18℃、1?50比例加晶种。去除标签后的Hsp B蛋白获得了分辨率1.8?的单晶。It was to obtain the monocrystal of Hsp B, the key monooxygenase in the nicotine degradation pathway from Pseudomonas 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 Hsp B with high purity. The optimal crystallization condition was identified as 22% PEG3350, 0.1 mol/L Bis-Tris p H6.5, 0.21 mol/L Mg Cl2, 18℃, seeding after orthogonal experiments. Hsp B with His-tag cleavaged can obtain a monocrystal which resolution reaches 1.8 ？.

关 键 词：恶臭假单胞菌 尼古丁代谢 单加氧酶 定点突变 结晶 

分 类 号：Q936[生物学—微生物学]