泛素链的体外制备、磷酸化修饰与标记方法  

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

作　　者：覃凌云 聂泽锋 唐淳 QIN Ling-Yun;NIE Ze-Feng;TANG Chun(Innovation Academy for Precision Measurement Science and Technology,Chinese Academy of Sciences,Wuhan 430071,China;College of Chemistry and Molecular Engineering,Peking University,Beijing 100871,China;University of Chinese Academy of Sciences,Beijing 100049,China)

机构地区：[1]中国科学院精密测量科学与技术创新研究院,武汉430071 [2]北京大学化学与分子工程学院,北京100871 [3]中国科学院大学,北京100049

出　　处：《生物化学与生物物理进展》2022年第8期1586-1596,共11页Progress In Biochemistry and Biophysics

摘　　要：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。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.

关 键 词：翻译后修饰 多聚泛素 磷酸化 去泛素化酶 分支型泛素 

分 类 号：Q5[生物学—生物化学] Q7