人表皮生长因子受体胞内酪氨酸激酶结构域原核表达

Prokaryotic Expression of the Intracellular Tyrosine Kinase Domain of Human Epidermal Growth Factor Receptor

目的研究人表皮生长因子受体(EGFR)胞内酪氨酸激酶结构域的原核表达及纯化条件。方法构建原核表达人EGFR胞内酪氨酸激酶结构域的重组质粒pGEX/GST-EGFR-TKD,在大肠杆菌中表达目的融合蛋白,通过在尿素中变性、在梯度尿素中透析复性,经GST融合蛋白纯化,肠激酶去除GST“标签”后获得人EGFR胞内酪氨酸激酶结构域蛋白。结果限制酶切分析和测序表明成功构建重组质粒pGEX/GST-EGFR-TKD;SDS-PAGE分析表明,异丙基硫代-β-D半乳糖苷诱导下可以表达融合蛋白GST-EGFR-TKD,但以不溶性包涵体的形式存在。经尿素变性、梯度透析复性及去除GST“标签”后,获得重折叠的人EGFR胞内酪氨酸激酶结构域蛋白。结论在大肠杆菌中可成功表达人EGFR胞内酪氨酸激酶结构域蛋白。

Objective To investigate the prokaryotie expression of the intraeellular tyrosine kinase domain of human epidermal growth factor receptor（EGFR） and the purification conditions. Methods Recombinant plasmid pGEX/GST-EGFR-TKD which contain human EGFR intracellular tyrosine kinase domain is constructed. The fusion protein was expressed in E. coli and then denatured by urea solution, and renewal with dialysis in gradient urea solution and purified. Enterokinase was added to remove GST- tag to get human EGFR-TKD. Results Recombinant plasmid pGEX/GST-EGFR-TKD was successfully constructed and confirmed by enzyme restriction and sequencing. SDS-PAGE identified fusion protein GST-EGFR-TKD was expressed under induction of IPTG in form of insoluble inclusion body. After denaturing in urea, gradient dialysis and cleavage of GST-tag, refolded human EGFR-TKD was obtained. Conclusion Human EGFR-TKD can be expressed in E. coll.