泛素化相关蛋白纯化及体外泛素化体系的建立

Purification of Ubiquitination-related Proteins and Establishment of Ubiquitination System in Vitro

为构建泛素化相关蛋白的纯化方法以及利用纯化出的蛋白进行体外泛素化反应,采用分子克隆技术构建出p ET-N-His-UBE1、p ET-N-His-CDC34-C-His和p ET-N-His-UBB-CHis载体,其中p ET-N-His-UBE1载体来源于Addgene公司。将构建好的原核表达载体转化到BL21大肠杆菌中进行诱导表达,利用蛋白纯化技术纯化出相应的蛋白;将纯化出的泛素化相关的蛋白按照一定的比例混合,在ATP存在的条件下发生泛素化反应。成功建立了蛋白纯化和体外泛素化体系,即在细菌中纯化出E1、E2和泛素,在ATP存在的情况下使泛素结合到E2上。该体系的构建能为更好地探索细胞内蛋白的相互作用及人类疾病的治疗提供更多有价值的依据。

Objective： this paper aims to establish the purification method of ubiquitin related protein and use the purified protein for in vitro ubiquitination. Methods： Using molecular cloning techniques to construct p ET-N-His-UBE1,p ET-N-His-CDC34-C-His and p ET-N-His-UBB-C-His,the p ET-N-His-UBE1 is obtained from the company. The prokaryotic expression vector was transformed into BL21 Escherichia coli to induce protein expression,and the protein purification technology was used to purify the target protein. The purified protein was mixed in a certain proportion,and the ubiquitination reaction occurred in the presence of ATP. Through the above series of methods,protein purification and in vitro ubiquitination systems were established. E1,E2 and ubiquitin were purified in Escherichia coli and ubiquitin was bound to E2 in the presence of ATP. Conclusion： The establishment of this system can provide a more valuable basis for us to explore the interaction of intracellular proteins and the treatment of human diseases.