植物学报 ›› 2019, Vol. 54 ›› Issue (6): 764-772.DOI: 10.11983/CBB19152

• 技术方法 • 上一篇    下一篇

植物蛋白的体外泛素化检测方法

赵庆臻1,刘利静2,谢旗3,4,于菲菲3,*()   

  1. 1 聊城大学生命科学学院, 聊城 252000
    2 山东大学生命科学学院, 青岛 266237
    3 中国科学院遗传与发育生物学研究所, 北京 100101
    4 中国科学院大学, 北京 100049
  • 收稿日期:2019-08-12 接受日期:2019-10-31 出版日期:2019-11-01 发布日期:2020-07-09
  • 通讯作者: 于菲菲
  • 基金资助:
    国家自然科学基金(No.31200907);山东省自然科学基金(No.ZR2014CL1009);植物基因组学国家重点实验室开放课题(No.2016A0219-14)

In Vitro Ubiquitination Assay for Plant Proteins

Qingzhen Zhao1,Lijing Liu2,Qi Xie3,4,Feifei Yu3,*()   

  1. 1 College of Life Sciences, Liaocheng University, Liaocheng 252000, China
    2 College of Life Sciences, Shandong University, Qingdao 266237, China
    3 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
    4 University of Chinese Academy of Sciences, Beijing 100049, China
  • Received:2019-08-12 Accepted:2019-10-31 Online:2019-11-01 Published:2020-07-09
  • Contact: Feifei Yu

摘要: 泛素激活酶(E1)、泛素耦联酶(E2)和泛素连接酶(E3)是蛋白质泛素化修饰的关键酶。在真核基因组上有大量基因编码这些泛素化相关的酶类或蛋白。检测这些泛素化修饰酶及其底物蛋白的生化特性和特异性是分析其生物学功能的重要内容。该文提供了一种简便快速检测体外泛素化反应的方法, 不仅可通过检测对DTT敏感的硫酯键的形成来判断E2的活性、检测E3的体外泛素化活性, 而且可以检测E2-E3和E3-底物的特异性。所用蛋白主要来源于拟南芥(Arabidopsis thaliana), 包括分属于绝大多数E2亚家族的成员, 可用于不同RING类型E3的活性检测。该方法不仅可以采用多种E2进行E3活性分析, 而且可以分析不同组合的E2-RING E3、RING E3-底物的泛素化活性等, 亦可应用于真核生物蛋白质尤其是植物蛋白的体外泛素化活性分析。

关键词: 体外泛素化, 泛素耦联酶(E2), 泛素连接酶(E3)

Abstract: Ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin protein ligase (E3) are the key enzymes of ubiquitin modification of substrate proteins. There are large amounts of genes encoding these ubiquitination enzymes in all eukaryotic genomes. Analyzing the biochemical characteristics and specificity of these enzymes and their substrate proteins is important for their functional study. Here we describe a simple and fast method for in vitro ubiquitination assay. In the presence of E1 and ubiquitin, E2 activity can be determined by detecting the DTT-sensitive thio-ester formation. The E3 activity of a putative protein as well as the E2-E3 or E3-substrate specificities can also be explored by in vitro ubiquitination assay. This system is mainiy based on proteins from Arabidopsis, which includes most varieties of Arabidopsis E2 proteins that are tested with several RING-finger type E3 ligases. This system facilitate not only the exploration of E3 activity in combination with various Arabidopsis E2 members but also the study of E2-RING E3 and RING E3-substrate specificities. This system is suitable for the ubiquitination assays of eukaryotic proteins, especially for plant proteins.

Key words: in vitro ubiquitination assay, ubiquitin conjugating enzyme (E2), ubiquitin protein ligase (E3)