Expression and purification of functional recombinant CUL2•RBX1 from E. coli.
Expression and purification of functional recombinant CUL2•RBX1 from E. coli.
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DOI：
10.1038/s41598-021-90770-x
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发表时间：
2021-05-27
影响因子：
4.6
通讯作者：
Liu X
中科院分区：
文献类型：
--
作者：
Diaz S;Li L;Wang K;Liu X
基于Cullin-2（CUL 2）的Cullin-RING连接酶（CRL 2）包括仅存在于多细胞生物体中的泛素E3连接酶家族，并且对于细胞过程如胚胎发生和病毒发病机制至关重要。CUL 2是结合可互换底物受体模块之一的支架蛋白，其由衔接蛋白和底物受体蛋白组成。VHL蛋白是已知靶向低氧诱导因子α（HIF 1 α）进行泛素化和降解的底物受体。由于其在重要细胞因子如HIF 1 α的泛素化中的关键作用，CRL 2已被研究其生物学功能和开发新的疾病治疗药物。鉴于CRL 2在生物学和生物医学研究中的重要性，有效生产功能性CUL 2蛋白的方法将极大地促进对CRL 2的机制和调控的研究。在这里，我们报告了两个成本有效的系统表达和纯化重组人CUL 2从大肠杆菌。coli细胞。纯化的CUL 2蛋白纯度约为95%，可以结合其底物受体模块，并且在体外测定中具有将泛素或泛素样蛋白转移到相应底物的酶促活性。所提出的方法学进展将有助于推进CRL 2功能和调控的研究。
Cullin-2 (CUL2) based cullin-RING ligases (CRL2s) comprise a family of ubiquitin E3 ligases that exist only in multi-cellular organisms and are crucial for cellular processes such as embryogenesis and viral pathogenesis. CUL2 is the scaffold protein that binds one of the interchangeable substrate receptor modules, which consists of adaptor proteins and the substrate receptor protein. The VHL protein is a substrate receptor known to target hypoxia-inducible factor α (HIF1α) for ubiquitination and degradation. Because of its critical role in the ubiquitination of important cellular factors such as HIF1α, CRL2s have been investigated for their biological functions and the development of novel therapeutics against diseases. Given the importance of CRL2s in biological and biomedical research, methods that efficiently produce functional CUL2 proteins will greatly facilitate studies on the mechanism and regulation of CRL2s. Here, we report two cost-effective systems for the expression and purification of recombinant human CUL2 from E. coli cells. The purified CUL2 proteins were ~ 95% pure, could bind their substrate receptor modules, and were enzymatically active in transferring ubiquitin or ubiquitin-like protein to the corresponding substrate in in vitro assays. The presented methodological advancements will help advance research in CRL2 function and regulation.
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影响因子：
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通讯作者：
Yang H
影响因子：
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通讯作者：
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