大肠杆菌ybfE基因的三种原核质粒表达水平的对比及蛋白纯化  

Comparison of expression levels of E. coli ybfE gene in three kinds of prokaryotic plasmids and the protein purification

作　　者：张树军[1,2] 狄建军[2] 

机构地区：[1]内蒙古大学生命科学学院,内蒙古呼和浩特010021 [2]内蒙古民族大学生命科学学院,内蒙古通辽028000

出　　处：《生物技术》2017年第4期337-341,382,共6页Biotechnology

摘　　要：[目的]构建大肠杆菌功能未知基因ybf E的pET16b、pET32a和p GEX-4T-1三种原核表达系统,通过对比表达水平筛选出最优表达体系,并纯化表达的可溶性Ybf E融合蛋白。[方法]使用pET16b、pET32a和p GEX-4T-1表达质粒构建pET16b-ybf E、pET32a-ybf E和p GEX-4T-1-ybf E原核表达载体,分别转化大肠杆菌BL21,IPTG诱导表达Ybf E融合蛋白,对三种表达系统的表达水平进行对比,并对pET16b-ybf E和pGEX-4T-1-ybf E表达体系的裂菌上清中的可溶性Ybf E融合蛋白液分别使用镍柱和GST蛋白纯化柱纯化。[结果]构建了pET16b-ybf E、pET32a-ybf E和p GEX-4T-1-ybf E原核表达体系,并使用IPTG诱导表达Ybf E融合蛋白。ybf E在p GEX-4T-1载体内的表达水平最高,接下来依次为pET16b和pET32a。pET16b-ybf E和p GEX-4T-1-ybf E表达的可溶性Ybf E融合蛋白纯化后浓度分别为86μg/m L和724μg/m L。[结论]成功构建了ybf E基因的三种原核表达系统,筛选出最佳表达体系,可溶性Ybf E融合蛋白得到纯化。[Objective] To construct three prokaryotic expression systems of ybf E gene by pET16 b,pET32 a and p GEX-4T-1,which is a function unknown gene of E. coli. Through comparing their expression levels to screen out the optimum expression system,and to purify the soluble Ybf E fusion protein. [Methods] Using pET16 b,pET32 a and p GEX-4T-1 expression plasmids to construct pET16b-ybf E,pET32a-ybf E and p GEX-4T-1-ybf E prokaryotic expression vectors,then respectively transformed into E. coli BL21,and were induced by IPTG to express Ybf E fusion protein. The expression level of three kinds of expression system was compared. The soluble Ybf E fusion protein expressed by pET16b-ybf E and p GEX-4T-1-ybf E expression systems in the supernatant after disintegrating bacteria was purified by nickel column and GST-Sefinose Kit respectively. [Results] The pET16b-ybf E,pET32a-ybf E and p GEX-4T-1-ybf E prokaryotic expression systems were constructed,and the Ybf E fusion proteins were expressed by IPTG. The expression level of ybf E in the p GEX-4T-1 was the highest,next was pET16 b and pET32 a in that order. The concentration of soluble Ybf E fusion protein expressed by pET16b-ybf E and p GEX-4T-1-ybf E expression system in the supernatant after disintegrating bacteria was 86 μg/m L and 724 μg/m L respectively. [Conclusion] Three prokaryotic expression systems of ybf E were successfully builded,and screened out the optimum expression system. The soluble Ybf E fusion proteins were purified.

关 键 词：ybf E基因 pET16b质粒 pET32a质粒 pGEX-4T-1质粒 蛋白纯化 

分 类 号：Q343.1[生物学—遗传学]