肿瘤抑制因子p53的泛素修饰被发现在多种细胞内生物过程中发挥关键调控作用。然而泛素化p53的获取困难限制了其生化机制研究的开展。本项目拟在前期工作基础上运用转肽酶Sort A催化的酶促合成方法实现性质均一的不同类型泛素化p53的化学合成，并以合成蛋白为工具重构体外生化研究体系，探索去泛素化酶特异性去泛素化的生物化学机制。具体内容为：a. 优化Sort A催化的蛋白酰肼酶促合成方法，高效获得C端酰肼的p53片段；b. 运用泛素链酶促合成及辅基介导的连接方法获得不同链长及连接类型泛素修饰的p53片段，并与p53酰肼片段进行连接获得性质均一的泛素修饰p53；c. 建立精准的体外生物化学及生物物理研究体系，探索去泛素化酶对泛素修饰p53的特异性水解机制。通过本项目的实施力求拓展蛋白质酶促合成方法体系，阐明泛素修饰p53的去泛素化机制，也将充分展示蛋白质化学合成在翻译后修饰蛋白研究中的重要价值。

Ubiquitination of the tumor suppressor p53 has recently been found to play a key regulatory role in a variety of intracellular biological processes. However, the difficulty for obtaining ubiquitinated p53 protein greatly limits its development in biochemistry and biophysics studies. This project plans to use the transpeptidase Sort A assisted protein semisynthesis method for the chemical synthesis of different types of ubiquitinated p53 with uniform structure and properties. The synthetic protein acts as a tool to accurately reconstruct in vitro biochemical research system, and then to explore biochemical processes and biophysical mechanisms of specific deubiquitination for the deubiquitinase CYLD. The specific plan is: a. Optimize Sort A mediated protein hydrazide synthesis method to efficiently obtain a C-terminal hydrazide-containing p53 fragment; b. Ubiquitin chain enzymatic synthesis system and auxiliary-mediated ligation strategy were used to obtain the C-terminal p53 fragments with different site, chain length. The full length ubiquitinated p53 can be obtained through hydrazide based native chamical ligation between the C-terminal p53 fragments with p53 hydrazide fragment; c. Using synthetic protein as a tool to establish a precise in vitro biochemistry and biophysical research system and explore the selective hydrolysis mechanism of deubiquitinase CYLD for the hydrolysis of different ubiquitinated p53 with different site, chain length. Through the implementation of this project, we try to expand the enzymatic assisted protein semi-synthesis method and elucidate the selective hydrolysis mechanism of deubiquitinase for ubiquitinated p53, and will also exhibit the important value of protein chemical synthesis in the study of post-translational modified proteins.