重组大鼠肾胆绿素还原酶在大肠杆菌中表达和纯化方法的研究  

Expression and purification of reductase rat kidney biliverdin recombinantfor rat kidney in E.coli.BL-21

作　　者：吕昌莲[1] 王秀宏[1] 周宏博[1] 申峰[1] 刘明[1] 尹珅[1] 周虹[1] 

机构地区：[1]哈尔滨医科大学生物化学与分子生物学教研室,黑龙江哈尔滨150086

出　　处：《哈尔滨医科大学学报》2003年第1期3-6,共4页Journal of Harbin Medical University

基　　金：国家自然科学基金资助项目 ( 3 0 0 0 0 0 3 0 ) ;黑龙江省科技厅重大项目 ( 10 5 11Z0 11)

摘　　要：目的　确定重组大鼠肾胆绿素还原酶在大肠杆菌中的表达条件及纯化方法。方法　将已构建的大鼠肾胆绿素还原酶 (BVR)的重组质粒pMW172a -BVR转入大肠杆菌BL - 2 1,分析不同温度、摇床转速对BVR表达的影响 ,确定可溶性表达最佳条件。使用超速离心、盐析、层析的方法纯化BVR。检测酶活性。结果　确定了BVR可溶性表达最佳条件为 37℃ (12 0± 5 )r/min ;确定了BVR具体纯化路线。所得蛋白纯度为 92 2 %、纯化倍数为 4 3倍、回收率为 19 8%的活性BVR蛋白。结论　获得了纯度高、具有活性的BVR蛋白 ,可作为工具酶用于某些相关酶的研究 。Objective To definite the optimal expression condition and purified methods of the recombinant of rat kidney biliverdin reductase in E.coli.Methods pMW172a-BVR,the recombinant of biliverdin reduetase (BVR)cDNA with plasmid,was transformed into E.coli BL-21.Through changing the temperature and shaking speed of culture bed respectively,analyzed their influences on BVR expression to definite the optimal expression condition.The purification process of expressed BVR from E.coli.BL-21 mainly included splitting bacterial with lysozyme and ultrasound,uhracentrifugation,salting out with ammonium sulfate and chromatog raphy.To keep the activity of BVR,the whole process was taken at 4℃.Results The optimal expression condition for soluble BVR was 370c(120±5)r/min;it provided fl feasible process for BVR purification.The result of G-100 chromatography Was single strap on SDS-PAGE and its purity Was 92.2%detected by scanning.the purification folds were 4.3 and the reclaim rate was 19.8%calculated according to the activity.Conclusion This research lay the foundation of studying the relationships between its structure and function,moreover,it could provide BVR for the research correlated enzymes.

关 键 词：肾胆绿素还原酶 大肠杆菌 表达条件 纯化方法 动物实验 

分 类 号：Q78[生物学—分子生物学]