氧化还原因子-1的原核表达纯化及其活性鉴定

Prokaryotic expression,purification and activity identification of redox factor 1

为建立氧化还原因子-1(Ref-1)的原核表达系统,将经过酶切后的人源Ref-1编码片段定向克隆到pGEX-4T-3载体上,构建了pGEX-4T-3/Ref-1原核表达载体,重组质粒转化至BL21(DE3)工程菌,异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导重组工程菌表达可溶性融合蛋白,谷胱甘肽琼脂糖凝胶4B(Glutathione Sepha-rose 4B)亲和纯化重组蛋白,透析后用聚乙二醇8000(PEG8000)浓缩,获得纯度达92%的融合蛋白,产率为3.7 mg/L,融合蛋白占菌体总蛋白的6.8%.蛋白质印迹(Western blot)显示该蛋白可以被抗体特异性识别,证实其为GST-Ref-1融合蛋白.GST-Ref-1蛋白体外增强了激活蛋白-1(AP-1)的DNA结合能力,并能拮抗H2O2造成的AP-1氧化损伤.

To construct the prokaryotic expression vector of redox factor-1（Ref-1）,the gene fragment encoding Ref-1 was inserted into pGEX-4T-3 prokaryotic expression vector.Recombinant plasmid was transformed into E.coli BL21（DE3） cells.After induction by IPTG,GST-Ref-1 fusion protein showed a high level expression in soluble form.The recombinant proteins were purified by affinity chromatography with Glutathione Sepharose 4B and concentrated by PEG8000.Purified GST-Ref-1 fusion protein accounted for 6.8% of total bacterial protein and the product yield was 3.7 mg/L.Its purity was over 92%.The recombinant proteins could react specifically with anti-Ref-1 antibody.GST-Ref-1 fusion protein enhanced the DNA binding activity of AP-1 and attenuated H2O2-mediated oxidative injury of AP-1.