SCF泛素连接酶的体外泛素化体系构建与检测
Constructing and detecting ubiquitination system of SCF E3 ligase in vitro
胡健健 1梅文聪 1张文慧 1刘主1
作者信息
- 1. 作物遗传改良全国重点实验室/湖北洪山实验室/华中农业大学生命科学技术学院,武汉 430070
- 折叠
摘要
为研究蛋白质的体外泛素化过程,利用大肠杆菌表达系统异源表达和纯化APPBP1/UBA3(E1)、UBC12(E2)、Cullin1-Rbx1(E3)和Nedd8(neural precursor cell expressed developmentally down-regulated pro-tein 8)蛋白,制备FITC-Cysteine 绿色荧光素标记的泛素Ub(Ubiquitin),构建了SCF泛素连接酶的体外泛素化体系,同时实现快速检测SCF泛素连接酶中Cullin1蛋白的自泛素化修饰.结果表明,建立的体外SCF E3自泛素化活性反应体系具有较高的可操作性和便利性.
Abstract
Covalent attachment of ubiquitin onto lysine residues of the substrate requires the coordinat-ed action of three classes of enzymes:the E1 ubiquitin-activating enzymes,the E2 ubiquitin-conjugating enzymes,and the E3 ubiquitin ligases.These ubiquitination-related proteins play a pivotal role in plant de-velopment and plant physiology.The APPBP1/UBA3(E1),UBC12(E2),Cullin1-Rbx1(E3)and Nedd8 were used to reconstitute SCF E3 ligase activity in vitro to study protein ubiquitination in vitro.Ub(Ubiquitin)labeled with FITC-Cysteine green fluorescein was prepared.The ubiquitination system of SCF ubiquitin ligase in vitro was constructed.The self-ubiquitination modification of Cullin1 protein in SCF ubiquitin ligase was rapidly detected.The results showed that the autoubiquitination active reaction system of SCF E3 in vitro established has high operability and convenience.
关键词
SCF泛素连接酶/泛素化修饰/Cullin1蛋白/荧光标记Key words
ubiquitination/SCF E3 ligase/Cullin1/fluorescence labeling引用本文复制引用
出版年
2024
NETL
NSTL
万方数据