转录因子E2F1蛋白纯化及甲基化鉴定  

Purification and Methylation Identification of Transcription Factor E2F1 Protein

作　　者：王颖[1] 邹世清[1] 陈波[1] 

机构地区：[1]武汉大学中南医院呼吸内科,湖北武汉430071

出　　处：《现代生物医学进展》2017年第21期4012-4015,共4页Progress in Modern Biomedicine

摘　　要：目的:构建人E2F1基因原核表达质粒p GEX-KG-E2F1,并在大肠杆菌中诱导表达。随后验证纯化得到的E2F1蛋白可作为底物被甲基化转移酶修饰。方法:构建原核表达质粒p GEX-KG-E2F1,在大肠杆菌BL-21中经异丙基硫代半乳糖苷(IPTG)诱导表达,利用GST亲和层析法纯化表达的E2F1蛋白。随后将纯化的E2F1蛋白作为底物,组蛋白甲基化转移酶SET7/9作为酶进行体外同位素标记放射自显影实验,检测纯化的E2F1蛋白能否被甲基化。结果:酶切鉴定和测序结果证明成功构建了原核表达载体p GEX-KG-E2F1,SDS-PAGE检测结果证明实现了人E2F1基因在大肠杆菌中的可溶性表达,放射自显影证明纯化得到的E2F1蛋白可作为底物被甲基化转移酶SET7/9甲基化。结论:成功构建了转录因子E2F1体外甲基化体系,为筛选新的能甲基化E2F1的酶奠定基础。Objective：To construct prokaryotic expression vector pGEX-KG-E2F1 of the human gene E2F1,and to express fusion protein and purify the E2F1 protein,and to validate that our purified E2F1 can be used as the substrate for histone methyltransferase-mediated methylation.Methods：The prokaryotic expression plasmid pGEX-KG-E2F1 was constructed and the expression of E2F1 was induced by IPTG in E.coli BL-21.And then we use GST affinity chromatography to purify E2F1 protein.Then we use the purified E2F1 protein as a substrate,and the histone methyltransferase enzyme SET7/9 as the enzyme for autoradiography experiments in vitro.Results：Enzyme digestion and sequencing results demonstrated the prokaryotic expression vector pGEX-KG-E2F1 was constructed successfully.SDS-PAGE results proved the E2F1 protein can be solublely expressed and autoradiography demonstrated that purified E2F1 protein can be methylated by histone methylation transfemse SET7/9.Conclusion：We successfully constructed E2F1 methylation system in vitro,and this work certainly laid a solid foundation for the further study of screening histone methyltransferases which can methylate E2F 1.

关 键 词：原核表达 组蛋白甲基化转移酶 E2F1 SET7/9 

分 类 号：R33[医药卫生—人体生理学]