Life Science Business Department, Shimadzu Corporation, Nishinokyo-Kuwabaracho, Nakagyo-ku, Kyoto, Japan.
J Biotechnol. 2010 Jan 1;145(1):73-8. doi: 10.1016/j.jbiotec.2009.10.009.
Ubiquitination is one of the most significant posttranslational modifications (PTMs). To evaluate the ability of an insect cell-free protein synthesis system to carry out ubiquitin (Ub) conjugation to in vitro translated proteins, poly-Ub chain formation was studied in an insect cell-free protein synthesis system. Poly-Ub was generated in the presence of Ub aldehyde (UA), a de-ubiquitinating enzyme inhibitor. In vitro ubiquitination of the p53 tumor suppressor protein was also analyzed, and p53 was poly-ubiquitinated when Ub, UA, and Mdm2, an E3 Ub ligase (E3) for p53, were added to the in vitro reaction mixture. These results suggest that the insect cell-free protein synthesis system contains enzymatic activities capable of carrying out ubiquitination. CBB-detectable ubiquitinated p53 was easily purified from the insect cell-free protein synthesis system, allowing analysis of the Ub-conjugated proteins by mass spectrometry (MS). Lys 305 of p53 was identified as one of the Ub acceptor sites using this strategy. Thus, we conclude that the insect cell-free protein synthesis system is a powerful tool for studying various PTMs of eukaryotic proteins including ubiqutination presented here.
泛素化是最重要的翻译后修饰（PTMs）之一。为了评估昆虫无细胞蛋白合成系统进行体外翻译蛋白泛素（Ub）缀合的能力，在昆虫无细胞蛋白合成系统中研究了多泛素链的形成。在 Ub 醛（UA）存在的情况下产生多泛素，UA 是一种去泛素化酶抑制剂。还分析了 p53 肿瘤抑制蛋白的体外泛素化，当 Ub、UA 和 Mdm2（E3 泛素连接酶（E3）用于 p53）添加到体外反应混合物中时，p53 被多泛素化。这些结果表明，昆虫无细胞蛋白合成系统包含能够进行泛素化的酶活性。从昆虫无细胞蛋白合成系统中很容易纯化出 CBB 可检测的泛素化 p53，从而可以通过质谱（MS）分析 Ub 缀合蛋白。使用这种策略鉴定了 p53 的 Lys305 是 Ub 受体位点之一。因此，我们得出结论，昆虫无细胞蛋白合成系统是研究包括此处介绍的泛素化在内的真核蛋白各种翻译后修饰的有力工具。
