油菜中—未知BnRCH蛋白的E3连接酶活性分析  

The E3 ligase activity analysis of an unknown BnRCH protein in Brassica napus

作　　者：陈存[1] 唐天元[1] 杨虹伟[1] 蔡黎[1] 杨毅[1] 李旭锋[1] 

机构地区：[1]四川大学生命科学学院与生物资源与生态环境教育部重点实验室,成都610064

出　　处：《四川大学学报（自然科学版）》2012年第5期1159-1163,共5页Journal of Sichuan University(Natural Science Edition)

基　　金：国家自然科学基金(30971816,30871322)

摘　　要：利用本实验室前期以ATP6为诱饵蛋白,通过酵母双杂交系统筛选到的一个N端含有RINGv结构(RING-variant)的蛋白,暂命名为BnRCH,构建GTK-BnRCH表达载体在原核细胞E.coli BL21(DE3)中高效表达GST-BnRCH融合蛋白,并将纯化的融合蛋白加入体外泛素反应体系,发现BnRCH在ATP、泛素、E1、E2存在的条件下,能催化多聚泛素化形成,具有E3连接酶活性.ATP6 was used as the bait protein to screen its interacting partners by yeast two-hybrid system. Then a fragment, which has a RINGv （RING-variant） structure, was therefore found, and we ten- tatively named it BnRCH. BnRCH from Brassica napus was cloned into the prokaryotic expression vector GTK to express BnRCH as a glutathione-s-transferase （GST） fusion protein. The recombinant ex- pression plasmid was then transformed into E. coli BL21 （DE3）. In the presence of human E1 and human E2, ubiquitination activity was observed in the presence of purified GST-BnRCH, indicating that BnRCH has E3 ligase activity in vitro.

关 键 词：BnRCH 原核表达 E3连接酶 

分 类 号：Q78[生物学—分子生物学]