抗癌蛋白NOR1基因工程重组蛋白表达条件的优化和纯化

目的:构建NOR1基因原核表达载体,并在大肠杆菌中原核表达NOR1重组蛋白,对其诱导条件进行优化,并纯化NOR1蛋白.方法:PCR扩增人NOR1基因全长开放阅读框,克隆入原核表达载体pET28b.重组质粒转化大肠杆菌,采用不同温度、不同抗生素浓度、不同IPTG浓度诱导NOR1蛋白表达.选择最佳条件大量诱导NOR1蛋白表...

Optimization of prokaryotic expression condition and purification of anti-cancer protein NOR1 in E.coli

Abstract：ObjectiveTo optimize the induction condition of human NOR1 gene expression in E.coli. and purify NOR1 recombinant proteins. MethodsA full-length cDNA of human NOR1 was inserted into the corresponding region of pET28b expression vector to yield recombinant prokaryotic expression vector pET28b-NOR1. The prokaryotic expression vector pET28b-NOR1 was introduced into the bacterial host E.coli Rosettablue(DE3). Recombinant NOR1 protein was induced at different conditions. Induction condition was optimized to obtain high yield of recombinant protein. At last, the recombinant NOR1 protein was purified by Ni-IDE chromatography resin. ResultsRecombinant NOR1 protein was induced by IPTG in a dose-dependent manner. Increase of kanamycin concentration and induction temperature resulted in high yield of recombinant protein. The most recombinant protein was found in inclusion bodies. The recombinant His-NOR1 protein was purified with Ni-IDE chromatography resin under denature condition. ConclusionIPTG, kanamycin concentration and temperature can affect the expression of recombinant NOR1 protein in pET28b system. High yield of recombinant NOR1 protein is achieved by inducing 1 mmol/L IPTG and 200 μg/mL kanamycin at 37 ℃. Recombinant His-NOR1 protein with high purity is purified.