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

作　　者：方迷 张敏敏 杨卓茹 陈婷[1] 张云龙[1] 陆昌瑞 Mi Fang;Minmin Zhang;Zhuoru Yang;Ting Chen;Yunlong Zhang;Changrui Lu(College of Chemistry,Chemical Engineering and Biotechnology,Donghua University,Shanghai 201620,China)

机构地区：[1]东华大学化学化工与生物工程学院

出　　处：《生物技术》2018年第6期520-525,共6页Biotechnology

基　　金：国家自然科学基金项目(“SAM核酸开关的结构与机理”,No.31300603)

摘　　要：[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.

关 键 词：人端粒酶 人端粒酶RNA 共表达 蛋白纯化 

分 类 号：Q784[生物学—分子生物学] Q785