人端粒酶与RNA模板的共表达及纯化

Co-expression and purification of the human telomerase with the RNA template

[目的]提高人端粒酶的表达量和可溶性,获得可溶性的人端粒酶。[方法]分别构建融合质粒pRT-ppSUMO和RNA质粒hTER-puc19,共同转化至大肠杆菌BL21(DE3)中,并优化共表达条件,通过亲和层析分离纯化得到人端粒酶。[结果]人端粒酶和RNA模板共表达体系将人端粒酶的表达水平提高了1.49倍;确定共表达中最佳IPTG诱导浓度为0.75mmol/L,最佳诱导温度为20℃,诱导时间为12~16h;经纯化最终得到浓度为29.61μg/mL,纯度为70%的人端粒酶。[结论]得到可溶性人端粒酶,可用于其晶体结构及功能的进一步研究。

[Objective]To improve the expression level and solubility of human telomerase,and obtain the soluble protein.[Methods]pRT-ppSUMO fusion plasmid and the hTER-puc19 RNA plasmid were separately constructed,and both co-transformed into E.coli BL21(DE3)cells,then the co-expression conditions were optimized and the target protein was purified by affinity chromatography.[Results]The co-expression system of the human telomerase with the RNA template increased human telomerase expression by 1.49 times;the optimum concentration of IPTG induction was 0.75 mmol /L and the optimum inducing temperature was 20℃ for 12-16 h;the resulting concentration of human telomerase after purification was 29.61 μg /mL,and the purity was 70%.[Conclusion]The soluble protein human telomerase was obtained,which could support further studies of its crystal structure and function.