大肠杆菌yfiF基因原核表达系统构建、表达条件优化及蛋白纯化  

Construction of the prokaryotic expression system of yfiF gene in E. coli,optimization of its induced expression condition and purification of the protein

作　　者：张树军[1,2] 狄建军[2] 张国文[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》2016年第3期229-233,共5页Biotechnology

摘　　要：[目的]构建大肠杆菌功能未知基因yfi F的原核表达系统,对诱导表达条件进行优化,并纯化表达的可溶性Yfi F融合蛋白。[方法]使用p ET16b表达质粒构建p ET16b-yfi F原核表达载体,转化大肠杆菌BL21,IPTG诱导表达Yfi F融合蛋白,对IPTG加入时机、终浓度及诱导时间进行优化,并使用镍柱纯化裂菌上清液中的可溶性Yfi F融合蛋白。[结果]构建了p ET16b-yfi F重组质粒,IPTG诱导表达Yfi F融合蛋白,最佳诱导条件为细菌生长至OD600值为1时加入IPTG,终浓度为0.1 mmol/L,诱导9 h。裂菌上清液中的可溶性Yfi F融合蛋白纯化后浓度为209μg/ml。[结论]成功构建了yfi F的原核表达系统,优化了诱导表达条件,可溶性Yfi F融合蛋白得到纯化。[ Objective] To construct the prokaryotic expression system of yfiF gene ,which is a function unknown gene of E. co- li, to optimize its induced expression condition, and to purify the soluble YfiF fusion protein. [ Methods] Using pET16b expres- sion plasmid to construct pET16b- yfiF prokaryotic expression vector, then transformed it into E. coli BI21, and was induced by IPTG to express fusion protein YfiF. The adding time, final concentration and induction time of IPTG was optimized. The sol- uble YfiF fusion protein in the supernatant after disintegrating bacteria was purified by nickel column. [ Results] The pET16b - yfiF recombinant plasmid was constructed, the YfiF fusion protein was expressed by IPTG, and the optimum induction conditions was that bacterial growth to OD6o0 value was 1.0 added IPTG,its final concentration was 0.1 mmol/L and induced for 9 h. The concentration of soluble YfiF fusion protein after disintegrating bacteria was 209 μg/ml. [ Conclusion] The prokaryotie expres- sion system of yfiF is successfully buiided, the induced condition is optimized, and the soluble yfiF fusion protein is purified.

关 键 词：yfiF基因 pET16b质粒 原核表达 蛋白纯化 

分 类 号：Q343.1[生物学—遗传学]