The State Key Laboratory Breeding Base of Bioresources and Eco-environments, Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Molecular Developmental Biology, School of Life Sciences, Southwest University, Beibei, Chongqing, China.
Department of Nephrology, Institute of Nephrology of Chongqing and Kidney Center of PLA, Xinqiao Hospital, Army Medical University, Chongqing, China.
Biosci Biotechnol Biochem. 2021 Apr 24;85(5):1140-1146. doi: 10.1093/bbb/zbab026.
Ubiquitination is involved in the regulation of numerous cellular functions. Research works in the ubiquitin realm rely heavily on ubiquitination assays in vitro and require large amounts of ubiquitin-activating enzyme (UBA1) and keep ATP supplies. However, UBA1 is hard to be obtained with large quantities using reported methods. We fused Escherichia coli adenylate kinase (adk) and mouse UBA1 and obtained fusion protein adk-mUBA1. The expression level of adk-mUBA1 increased about 8-fold compared with mUBA1 in an E. coli expression system, and adk-mUBA1 was easily purified to 90% purity via 2 purification steps. The purified adk-mUBA1 protein was functional for ubiquitination and could use ATP in addition to ADP as energy supply and had a higher catalytic activity than mUBA1 in cell lysis. adk-mUBA1 can be applied to preparing ubiquitin-modified substrates and kinds of ubiquitin chains in a chemical synthesis process and is a preferable application than mUBA1 in vitro ubiquitination.
泛素化参与了许多细胞功能的调节。在泛素领域的研究工作严重依赖于体外的泛素化测定，需要大量的泛素激活酶（UBA1）和保持 ATP 的供应。然而，使用已报道的方法很难获得大量的 UBA1。我们融合了大肠杆菌腺苷酸激酶（adk）和小鼠 UBA1，得到了融合蛋白 adk-mUBA1。与大肠杆菌表达系统中的 mUBA1 相比，adk-mUBA1 的表达水平增加了约 8 倍，并且 adk-mUBA1 可以通过 2 步纯化达到 90%的纯度。纯化的 adk-mUBA1 蛋白具有泛素化功能，除了 ADP 之外，还可以使用 ATP 作为能量供应，并且在细胞裂解物中的催化活性比 mUBA1 更高。adk-mUBA1 可应用于化学合成过程中制备泛素修饰的底物和各种泛素链，并且比体外泛素化中的 mUBA1 更具应用前景。
