ASCT2胞外域ECL2原核可溶性表达条件的优化及其纯化

目的 构建重组人ASCT2胞外域ECL2融合蛋白的原核表达载体,优化其在大肠埃希菌中可溶性表达的条件,并获得纯化的ECL2蛋白.方法 以PCR方法扩增编码ECL2的DNA片段,插入至pET-41b载体中,构建ECL2的原核表达载体,转化至大肠埃希菌,优化可溶性表达条件,GSH-Agarose亲和层析纯化并鉴定,与MCF-7细胞结合活性的测定.结果 免疫印迹显示,ECL2融合蛋白既表达于包涵体中,也能以可溶性形式表达.随IPTG浓度的增高,可溶性表达水平提高;培养温度为28 ℃和33 ℃时可溶性产物高于37 ℃;而随诱导时间的延长至12 h以上,可溶性表达量下降.可溶性表达优化条件为:温度33 ℃、IPTG浓度0.4 mmol/L、诱导时间4 h.经亲和层析获得高纯度ECL2蛋白并具有与MCF-7细胞结合活性.结论 优化了ECL2融合蛋白的可溶性表达条件,通过亲和层析一步法可获得高纯度融合蛋白.

Optimization of Prokaryotic Expression and Purification of Soluble ASCT2 Extracellular Domain ECL2

Objective To construct prokaryotic expression vector containing the extracellular domain ECL2 of human ASCT2 and to optimize expression condition for soluble pure ECL2 in Escherichia coli(E. coli).Methods DNA fragment encoding ECL2 was amplified by PCR and inserted into pET-41b prokaryotic expression vector. The obtained ECL2 expressing vector was transformed into E. coli and the soluble expression condition was optimized. GSH-Agarose column was used to purify ECL2 protein. Binding activity in MCF-7 cell was tested.Results Immunoblotting showed that ECL2 fusion protein was expressed both in inclusion body and in soluble form. Soluble expression level was higher at 28 ℃ and 33 ℃than at 37 ℃and increased with the IPTG concentration,but decreased with the time prolongation. The optimized condition was determined as 0.4 mmol/L IPTG and 4 h induction at 33 ℃. By use of affinity chromatography,highly pure ECL2 protein was obtained,showing binding activity in MCF-7 cells as tested.Conclusion The condition for soluble ECL2 expression was optimized and the fusion protein was purified by one step affinity column.