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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.
In this experiment, a protein with RING-variant at the N-terminus selected by yeast two-hybrid system was tentatively named as BnRCH by using ATP6 as the bait protein in the pre-laboratory, and the GTK-BnRCH expression vector was constructed in prokaryocyte E. coli GST-BnRCH fusion protein was highly expressed in BL21 (DE3), and the purified fusion protein was added to the ubiquitin reaction system. It was found that BnRCH can catalyze polyubiquitination formation in the presence of ATP, ubiquitin, E1 and E2 , With E3 ligase activity.