泛素链的体外制备、磷酸化修饰与标记方法

目的 为了制备不同链种类、不同链长及磷酸化修饰的泛素样品。方法 本文主要以生物酶法为手段对以上样品的制备路线进行阐述。制备的主要方法分为两种，一是采用逐次添加的方式达到泛素链延长的目的，二是通过一次酶反应制备混合的多聚泛素链，然后对不同链长的泛素链进行纯化分离。结果 以上两种策略都能达到制备多聚泛素链的目的。进一步，通过对泛素进行磷酸化修饰，制备了磷酸化的泛素样品。通过K11和K48的泛素酶制备了K11/K48分支链泛素。结论 基于以上泛素链的制备路线，可以进一步对不同链接形式的不同亚基进行磷酸化修饰等翻译后修饰，也可以通过在特定亚基进行同位素标记及在特定位点引入小分子探针，进而进行NMR和FRET的测定。综上所述，本方法将为从事泛素信号通路和泛素生化研究的科学家提供借鉴和帮助。

In Vitro Preparation of Ubiquitin Chain and Its Phosphorylation Modification and Labeling Sample

Objective In order to prepare ubiquitin samples with different chain types, different chain lengths and phosphorylation modifications.Methods This study mainly uses the biological enzymatic method to describe the preparation routes of the above samples. There are two main methods, the first method is to add ubiquitin monomer one by one and then extend the ubiquitin chain; the second is to prepare mixed polyubiquitin chains through an enzymatic reaction, and then purify and separate these mixed chains.Results The experimental results show that both methods can achieve the purpose of preparing polyubiquitin chains. Further, phosphorylated ubiquitin samples were prepared by phosphorylation of ubiquitin. K11/K48 branched chain ubiquitin was prepared by K11 and K48 ubiquitinase.Conclusion In short, based on the above preparation route of the ubiquitin chain, it is possible to further perform post-translational modifications such as phosphorylation modification on different subunits of different link forms. The site-specific attachment of a prosthetic probe and isotope labeling on different subunits allowed us to perform NMR and FRET measurements. In summary, these methods presented here can provide reference and help for scientists involved in Ub research.