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
发布时间：2018-09-02 06:01
【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。
[Abstract]:[objective] to construct three prokaryotic expression systems of E. coli function unknown gene ybf E, pET16b,pET32a and p GEX-4T-1, and to select the optimal expression system by comparing the expression level, and to purify the expressed soluble Ybf E fusion protein. [methods] the prokaryotic expression vectors of pET16b-ybf pET32a-ybf E and p GEX-4T-1-ybf E were constructed by using pET16b,pET32a and p GEX-4T-1 expression plasmids. The expression levels of the three expression systems were compared with those of E. coli BL21,IPTG induced expression of Ybf E fusion protein. The soluble Ybf E fusion protein in the supernatant of pET16b-ybf E and pGEX-4T-1-ybf E expression system was purified by nickel column and GST protein column, respectively. [results] the prokaryotic expression systems of pET16b-ybf pET32a-ybf E and p GEX-4T-1-ybf E were constructed, and the expression level of Ybf E fusion protein. Ybf E was the highest in p GEX-4T-1 vector. The concentration of soluble Ybf E fusion protein expressed by pET16b, pET32a.pET16b-ybf E and p GEX-4T-1-ybf E was 86 渭 g / mL and 724 渭 g / mL, respectively. [conclusion] three prokaryotic expression systems of ybf E gene were successfully constructed, and the best expression system was selected. The soluble Ybf E fusion protein was purified.
【作者单位】： 内蒙古大学生命科学学院;内蒙古民族大学生命科学学院;
【分类号】：Q78
[Abstract]:[objective] to construct three prokaryotic expression systems of E. coli function unknown gene ybf E, pET16b,pET32a and p GEX-4T-1, and to select the optimal expression system by comparing the expression level, and to purify the expressed soluble Ybf E fusion protein. [methods] the prokaryotic expression vectors of pET16b-ybf pET32a-ybf E and p GEX-4T-1-ybf E were constructed by using pET16b,pET32a and p GEX-4T-1 expression plasmids. The expression levels of the three expression systems were compared with those of E. coli BL21,IPTG induced expression of Ybf E fusion protein. The soluble Ybf E fusion protein in the supernatant of pET16b-ybf E and pGEX-4T-1-ybf E expression system was purified by nickel column and GST protein column, respectively. [results] the prokaryotic expression systems of pET16b-ybf pET32a-ybf E and p GEX-4T-1-ybf E were constructed, and the expression level of Ybf E fusion protein. Ybf E was the highest in p GEX-4T-1 vector. The concentration of soluble Ybf E fusion protein expressed by pET16b, pET32a.pET16b-ybf E and p GEX-4T-1-ybf E was 86 渭 g / mL and 724 渭 g / mL, respectively. [conclusion] three prokaryotic expression systems of ybf E gene were successfully constructed, and the best expression system was selected. The soluble Ybf E fusion protein was purified.
【作者单位】： 内蒙古大学生命科学学院;内蒙古民族大学生命科学学院;
【分类号】：Q78
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