Click chemistry for targeted protein ubiquitylation and ubiquitin chain formation
Click chemistry for targeted protein ubiquitylation and ubiquitin chain formation
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DOI：
10.1038/nprot.2015.106
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发表时间：
2015-10-01
期刊：
影响因子：
14.8
通讯作者：
Marx, Andreas
中科院分区：
文献类型：
--
作者：
Roesner, Daniel;Schneider, Tatjana;Marx, Andreas
在这里，我们描述了一个简单的协议，使用点击化学有效地产生位点特异性的泛素-蛋白质缀合物。通过使用两种不同的方法来扩展遗传密码，将Cu-I催化的叠氮化物-炔环加成（CuAAC）所必需的两个生物正交官能团（炔和叠氮化物）与非天然氨基酸共选择性地掺入到感兴趣的蛋白质中。随后通过CuAAC在体外用纯化的蛋白质进行蛋白质泛素化。此外，我们提供了一个方案，用于将两个非天然氨基酸掺入到一个单一的泛素中，产生一个“双功能”蛋白质，该蛋白质含有一个炔和一个叠氮化物功能，从而能够组装的自由泛素链以及泛素链缀合到靶蛋白。我们的程序能够在1周内合成不可水解的泛素-蛋白质缀合物（考虑到相关的cDNA在手边），并且它从1升表达培养物中产生毫克量的缀合物。本文所述的方法比其他方法更快且更省力，并且它仅需要标准分子生物学设备。此外，该方案可以容易地适应于在任何靶蛋白的任何位点实现缀合，这有助于产生定制的泛素-蛋白质缀合物。
Herein we describe a simple protocol for the efficient generation of site-specific ubiquitin-protein conjugates using click chemistry. By using two different methods to expand the genetic code, the two bio-orthogonal functionalities that are necessary for Cu-I-catalyzed azide-alkyne cycloaddition (CuAAC), an alkyne and an azide, are co-translationally incorporated into the proteins of interest with unnatural amino acids. Protein ubiquitylation is subsequently carried out with the purified proteins in vitro by CuAAC. In addition, we provide a protocol for the incorporation of two unnatural amino acids into a single ubiquitin, resulting in a 'bifunctional' protein that contains both an alkyne and an azide functionality, thereby enabling assembly of free ubiquitin chains as well as ubiquitin chains conjugated to a target protein. Our procedure enables the synthesis of nonhydrolyzable ubiquitin-protein conjugates within 1 week (given that the relevant cDNAs are at hand), and it yields conjugates in milligram quantities from 1-liter expression cultures. The approach described herein is faster and less laborious than other methods, and it requires only standard molecular biology equipment. Moreover, the protocol can be readily adapted to achieve conjugation at any site of any target protein, which facilitates the generation of custom-tailored ubiquitin-protein conjugates.