人HER2胞外区基因重组及重组蛋白的纯化

Recombination of Human HER2 Gene Encoding the Extracellular Domain and Purification of Fusion Protein

为在体外纯化可溶性HER 2胞外区蛋白并提高其原核表达产量,将多聚酶链式反应(PCR)方法获得的HER 2基因胞外段编码区重组至pGEX-6P-1质粒,转化大肠杆菌BL 21,采用不同的诱导条件分析融合蛋白的表达及其溶解性,不溶的包涵体经变性复性,与裂解液上清分别经G lu tath ione Sepharose 4B亲合纯化融合蛋白。结果发现,重组蛋白在原核细胞中以可溶和不可溶两种表达形式存在,但以不溶的包涵体形式为主。低温(30℃)、较高的细菌密度(A 600=1.8)和适当的培养基添加剂能有利于可溶性融合蛋白的表达。G lu tath ione Sepharose 4B亲合纯化细菌裂解液上清和复性后的融合蛋白,其产量为1.23 m g/L菌液,最大限度地获得了可溶性的HER 2蛋白胞外段,为HER 2特异性抗体的制备及其功能研究打下了良好的基础。

To purify the extracellular domain of HER2 in vitro and improve its prokaryotic expression abundance, the cDNA fragment encoding extracellular domain of HER2 was obtained by PCR and cloned into the expression vector pGEX-6P-1. After transforming it into Escherichia coli BL21, we instituted an investigation of different inducing conditions to try out the optimal condition for expressing soluble fusion protein. As for insoluble inclusion bodies, they were dissolved in 8 M Urea and refolded in refolding buffer. The soluble protein and the refolded protein were purified with Glutathione Sepharose 4B, respectively. The results showed that both the soluble and insoluble protein existed in Escherichia coli, but the majority was insoluble. It is beneficial to the expression of soluble fusion protein by induction at lower temperature （30℃） and higher optical density （A600= 1.8） with the use of certain additive in medium. By purification of the supernatant of the lysate and refolded protein, the yield of the fusion protein was about 1.23 mg per liter culture. As a result, we have obtained the maximum soluble extracellular domain of HER2 protein,and thus have laid a foundation for further work on functional study and antibody preparation for HER2.