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

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.