TRAF6-GST融合蛋白在大肠杆菌中的表达及纯化

Expression in Escherichia coli and Purification of GST tag TRAF6

目的:在大肠杆菌中表达肿瘤坏死因子受体相关因子6(TRAF6)与GST的融合蛋白并进行纯化。方法:采用PCR方法从肝文库中扩增编码TRAF6的DNA片段,将其插入原核表达载体pGEX-4T-2,构建GST-TRAF6原核表达载体,并转入大肠杆菌BL21(DE3)中,用IPTG诱导表达;用谷胱甘肽-琼脂糖珠亲和纯化表达的GST-TRAF6融合蛋白。结果:酶切鉴定和测序分析显示,长为1569 bp的TRAF6 DNA片段在pGEX-4T-2-TRAF6中的碱基序列、插入位点及读框正确,且位于表达载体的GST序列下游;经IPTG最佳浓度0.5 mmol/L诱导表达、亲和纯化后,获得了相对分子质量约85×103的GST-TRAF6融合蛋白。结论:构建了重组GST-TRAF6原核表达载体,获得了GST-TRAF6的大肠杆菌BL21表达菌株及GST-TRAF6融合蛋白,利于深入研究TRAF6的功能。

Objective： To express tumor necrosis factor receptor associated factor-6（TRAF6） fused with GST tag in E.coli BL21（DE3） and to purify it.Methods： The gene of TRAF-6 was amplified from liver gene library by PCR method,and was inserted into prokaryotic expression plasmid pGEX-4T-2 to construct prokaryotic expression vector of GST-TRAF6.Induced by IPTG,the recombinant GST-TRAF6 was expressed.At last,we purified recom-binant protein of GST-TRAF6 with glutathione-agarose beans.Results： Recombinant expression plasmid of GST-TRAF6 with the TRAF6 DNA fragment length of 1569 bp was confirmed by restriction enzyme digestion and se-quencing.The optimal IPTG concentration was 0.5 mmol / L determined by pretest.TRAF6 was downstream of GST gene,so we could obtain TRAF6 protein about 85 kD by affinity purification.Conclusion： Recombinant prokaryotic expression vector of GST-TRAF6 was correctly constructed,and the purified recombinant protein of GST-TRAF6 was obtained.