School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, 210023, Jiangsu, China.
The Drug Research Center of Immunological Diseases, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China.
Anal Biochem. 2023 Mar 1;664:115044. doi: 10.1016/j.ab.2023.115044. Epub 2023 Jan 12.
As one of the most prevalent protein post-translational modifications, ubiquitin modification plays a momentous role in regulating varied cellular functions. Different polyubiquitin linkage types have diverse effects on cell signaling. However, compared with short ubiquitin chains, the preparation of long ubiquitin chains remains difficult and expensive to purchase commercially. In this study, we constructed an enzyme library of ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin-ligase E3, which are specific for synthesizing K63, K48, and M1 linked polyubiquitin chains. We demonstrate that these distinctly linked polyubiquitin chains could be synthesized and purified with high yield and purity. More importantly, this method can synthesize longer ubiquitin chains, the longest can reach more than fifteen ubiquitin molecules, which provides great convenience for ubiquitin-related structural and functional studies.
作为最普遍的蛋白质翻译后修饰之一，泛素修饰在调节各种细胞功能方面起着重要作用。不同的多泛素连接类型对细胞信号转导有不同的影响。然而，与短泛素链相比，长泛素链的制备仍然难以实现，并且商业上购买也很昂贵。在这项研究中，我们构建了一个泛素激活酶 E1、泛素结合酶 E2 和泛素连接酶 E3 的酶文库，这些酶专门用于合成 K63、K48 和 M1 连接的多泛素链。我们证明了这些不同连接的多泛素链可以以高产率和高纯度进行合成和纯化。更重要的是，这种方法可以合成更长的泛素链，最长可达十五个以上的泛素分子，为泛素相关结构和功能研究提供了极大的便利。
