融合蛋白Trx-T4 DL可溶性表达、纯化与其生物活性应用

构建含SUMO、IF2、GST、NusA、MsyB、Trx和MBP融合标签的重组表达载体,转化到大肠杆菌E.coli Transetta (DE3)中进行自诱导(auto-induction,AI)表达,以提高T4 DNA连接酶(T4 DL)的表达产量。通过磁珠法检测融合蛋白的可溶性表达情况,10% SDS-PAGE电泳结果显示,重组菌Transetta (DE3)(pNBEVⅡ-T4 DL)诱导表达的可溶性融合蛋白Trx-T4 DL的产量最多,经诱导培养条件优化后,Trx-T4 DL的可溶性大幅度提高,确定了最佳诱导条件为30℃、装瓶量50 mL/250 mL、接种量2‰、pH7。分别用镍柱和MagNi磁珠纯化重组菌破碎后上清中的融合蛋白Trx-T4 DL,结果显示后者纯化效率更高,最终获得的融合蛋白浓度为1700.462 mg/L。与其他公司T4 DL活性进行比较,检测其酶活性约为500 U/μL,并使其成功应用于低背景重组克隆载体构建中,为融合蛋白Trx-T4 DL的生产及应用提供理论基础。

Soluble expression,purification and bioactive applications of recombination fusion protein Trx-T4 DL

The expression vector was constructed by plasmids containing SUMO,IF2,GST,NusA,MsyB,Trx and MBP fusion tags,which was transformed into E.coli Transetta(DE3) strain,to improve the expression yield of T4 DNA ligase(T4 DL).Then they were induced expression by auto-induction. The soluble expression of the fusion protein was efficiently and accurately detected by magnetic beads.The results showed that the yield of soluble fusion protein Trx-T4 DL induced expression from recombinant bacteria Transetta(DE3) (pNBE VⅡ-T4 DL) was the highest.Its solubility was greatly improved after the culture conditions were optimized,and the optimal induction conditions were 30℃,bottling volume 50 mL/250 mL,inoculation amount 2%,pH7. Meanwhile, the soluble T4 DL in the supernatant after disintegrating recombinant bacteria was purified by Ni+ column and MagNi magnetic beads,respectively.The high purity T4 DL was efficiently purified by MagNi magnetic beads,and its concentration was 1700.462 mg/L.Comparing with T4 DL activity of other companies,the enzymatic activity was determined as 500 U/mL, then it was successfully applied to the construction of low background cloning vector. It could provide a theoretical basis for the production and application of fusion protein Trx-T4 DL.