| Autor: |
Qingyun Zheng, Tian Wang, Junxiong Mao, Guochao Chu, Lujun Liang, Yangwode Jing, Chong Zuo, Yuanyuan Yu, Honggang Hu, Man Pan |
| Rok vydání: |
2021 |
| Předmět: |
|
| Zdroj: |
Nature protocols. |
| ISSN: |
1750-2799 |
| Popis: |
Ubiquitination regulates almost every life process of eukaryotes. The study of the ubiquitin (Ub) coupling or decoupling process and the interaction study of Ub-Ub binding protein have always been the central focus. However, such studies are challenging, owing to the transient nature of Ub-coupling enzymes and deubiquitinases in the reactions, as well as the difficulty in preparing large quantities of polyubiquitinated samples. Here we describe a recently developed strategy for the efficient preparation of analogs of Ub chains and analogs for Ub coupling and uncoupling intermediates, which facilitate the study of the ubiquitination process. The strategy includes mainly the following steps: (i) the bifunctional molecule conjugation on the only cysteine (Cys) residue of a target protein (usually a Ub or Ub-conjugating enzyme), exposing an orthogonal reactive site for native chemical ligation; (ii) covalent ligation with a Ub-derived thioester, exposing a free sulfhydryl; and (iii) (optional) a disulfide bond formation with a substrate protein (mainly with only one Cys protein) through nonactivity-based cross-linking or with a deubiquitinase (mainly with several Cys residues) through activity-based cross-linking. When the bifunctional molecule and target proteins are obtained, the final products can be prepared in milligram quantities within 2 weeks. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
|
Full text from SpringerLink