食品工业科技2018，Vol.39Issue(7)：83-89,96,8.DOI:10.13386/j.issn1002-0306.2018.07.017
融合蛋白Trx-T4DL可溶性表达、纯化与其生物活性应用
Soluble expression, purification and bioactive applications of recombination fusion protein Trx-T4 DL
摘要
Abstract
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 VⅦ-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.关键词
T4 DNA连接酶/融合标签/自诱导/磁珠法/纯化/低背景克隆载体/连接Key words
T4 DNA ligase/fusion tags/auto-induction/magnetic bead method/purification/low background cloning vector/ligation分类
轻工纺织引用本文复制引用
付大伟,陈启蒙,徐伟..融合蛋白Trx-T4DL可溶性表达、纯化与其生物活性应用[J].食品工业科技,2018,39(7):83-89,96,8.基金项目
哈尔滨商业大学博士科研启动基金项目(92508633). （92508633）
