A simple and high-sensitivity method for analysis of ubiquitination and polyubiquitination based on wheat cell-free protein synthesis
A simple and high-sensitivity method for analysis of ubiquitination and polyubiquitination based on wheat cell-free protein synthesis
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DOI：
10.1186/1471-2229-9-39
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发表时间：
2009-04-06
影响因子：
5.3
通讯作者：
Sawasaki, Tatsuya
中科院分区：
文献类型：
--
作者：
Takahashi, Hirotaka;Nozawa, Akira;Sawasaki, Tatsuya
背景资料：泛素化由至少三种酶的连续作用介导：E1（泛素活化酶），E2（泛素缀合酶）和E3（泛素连接酶）蛋白。靶蛋白的多泛素化也涉及几个关键的细胞过程。虽然拟南芥基因组研究估计有超过1,300种蛋白质参与泛素化，但对这些蛋白质的生化功能知之甚少。在这里，我们展示了一种新的，简单的和高灵敏度的方法，在体外分析泛素化和聚泛素化的基础上小麦无细胞蛋白合成和发光detection.Results：使用小麦无细胞合成，11个E3蛋白拟南芥全长cDNA模板。在翻译混合物或来自翻译混合物的纯化重组蛋白中分析这些蛋白。在我们使用FLAG-或His-标记的和生物素化的泛素的发光方法中，检测到AtUBC 22、UPL 5和UPL 7（HECT）和CIP 8（RING）上的聚泛素链。此外，使用生物素化的泛素和FLAG标记的重组蛋白检测泛素与这些蛋白的结合。此外，RING 6亚组的筛选表明At 1g 55530能够像CIP 8一样形成多聚泛素链。有趣的是，这些泛素化进行没有添加外源E1和/或E2蛋白，表明这些酶是内源性的小麦无细胞系统。粗翻译反应混合物中多聚遍在蛋白质的含量不受MG 132处理的影响，表明我们的系统不含26 S蛋白酶体依赖的蛋白质降解活性。结论：在这项研究中，我们开发了一种简单的基于小麦无细胞的发光方法，可以成为全面的遍在蛋白化分析的有力工具。
Background: Ubiquitination is mediated by the sequential action of at least three enzymes: the E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase) proteins. Polyubiquitination of target proteins is also implicated in several critical cellular processes. Although Arabidopsis genome research has estimated more than 1,300 proteins involved in ubiquitination, little is known about the biochemical functions of these proteins. Here we demonstrate a novel, simple and high-sensitive method for in vitro analysis of ubiquitination and polyubiquitination based on wheat cell-free protein synthesis and luminescent detection.Results: Using wheat cell-free synthesis, 11 E3 proteins from Arabidopsis full-length cDNA templates were produced. These proteins were analyzed either in the translation mixture or purified recombinant protein from the translation mixture. In our luminescent method using FLAG- or His-tagged and biotinylated ubiquitins, the polyubiquitin chain on AtUBC22, UPL5 and UPL7 (HECT) and CIP8 (RING) was detected. Also, binding of ubiquitin to these proteins was detected using biotinylated ubiquitin and FLAG- tagged recombinant protein. Furthermore, screening of the RING 6 subgroup demonstrated that At1g55530 was capable of polyubiquitin chain formation like CIP8. Interestingly, these ubiquitinations were carried out without the addition of exogenous E1 and/or E2 proteins, indicating that these enzymes were endogenous to the wheat cell-free system. The amount of polyubiquitinated proteins in the crude translation reaction mixture was unaffected by treatment with MG132, suggesting that our system does not contain 26S proteasome-dependent protein degradation activity.Conclusion: In this study, we developed a simple wheat cell-free based luminescence method that could be a powerful tool for comprehensive ubiquitination analysis.