多聚泛素及类泛素蛋白的化学合成研究

发布时间：2018-04-20 14:25

本文选题：泛素、类泛素蛋白 + 蛋白质化学合成　；参考：《合肥工业大学》2017年硕士论文

【摘要】：蛋白质的泛素化及类泛素化修饰在真核生物细胞内广泛存在并参与了大部分重要的细胞生命过程,包括信号传导、DNA损伤修复、蛋白降解等。不同底物的泛素及类泛素化修饰类型多种多样且具有不同的功能,保证了修饰功能的多样性和特异性。目前有相当一部分具有重要功能的泛素及类泛素蛋白由于缺乏特异性的酶促合成系统而不易获取。同时,获取纯净均一的泛素探针用于研究去泛素化酶的水解机制更为困难。蛋白质化学合成技术凭借其能够在原子尺度精准构筑蛋白质的优势,在传统生物酶法获取泛素、类泛素蛋白存在制约的情况下,提供了一种行之有效的策略。本文研究工作以蛋白质化学合成技术为工具,围绕泛素及类泛素蛋白的化学合成开展研究。首先通过使用本课题组发展的一锅法两片段连接-脱硫策略,我们高效地合成了类泛素化蛋白NEDD8以及泛素探针Ub-AMC,与前人方法相比合成步骤更少,效率更高。此后,作者又运用基于非天然氨基酸定点嵌入的蛋白质半合成方法对多聚泛素链的合成进行了探索。我们首先通过嵌入Alloc-Lys并使用Boc保护其他赖氨酸侧链氨基的方法成功制备了可用于连接的泛素片段;此后运用辅基介导的自然化学连接反应来实现泛素片段间异肽键的高效制备。本研究工作中成功制备的赖氨酸48位(K48)形成异肽键的二泛素验证了上述策略的可行性。本工作为进一步化学合成其他位点的多聚泛素、类泛素链提供了一种普适性方法,进而为阐明蛋白质泛素、类泛素化修饰的特异性识别、降解机制提供基础。
[Abstract]:Protein ubiquitization and ubiquitin modification exist widely in eukaryotic cells and participate in most important cellular life processes, including signal transduction DNA damage repair, protein degradation and so on. The types of ubiquitin and ubiquitin modified by different substrates are various and have different functions, which ensures the diversity and specificity of the modification functions. At present, a considerable number of ubiquitin and ubiquitin like proteins with important functions are difficult to obtain due to the lack of specific enzymatic synthesis system. At the same time, it is more difficult to obtain pure and uniform ubiquitin probe to study the hydrolysis mechanism of diubiquitin. Because protein chemical synthesis technology has the advantage of accurately constructing proteins on atomic scale, it provides an effective strategy for obtaining ubiquitin by traditional biological enzyme method and the restriction of ubiquitin protein. In this paper, the chemical synthesis of ubiquitin and ubiquitin protein was studied by using protein chemical synthesis technology as a tool. Firstly, we synthesized ubiquitin NEDD8 and Ub-AMC efficiently by using the one-pot two-fragment connection-desulphurization strategy developed by our team. Compared with previous methods, we synthesized Ub-AMCs with less steps and higher efficiency. Since then, the author has explored the synthesis of polyubiquitin chain by protein semi-synthesis method based on non-natural amino acid fixed point intercalation. Ubiquitin fragments were successfully prepared by embedding Alloc-Lys and protecting other lysine side chain amino groups by Boc, and then the heteropeptide bonds between Ubiquitin fragments were efficiently prepared by natural chemical bonding mediated by auxiliary groups. The diubiquitin of isopeptide bond formed by Lysine 48 K48 was proved to be feasible. This work provides a universal method for the further chemical synthesis of polyubiquitin and ubiquitin chain at other sites and provides a basis for elucidating the specific recognition and degradation mechanism of protein ubiquitin and ubiquitin modification.
【学位授予单位】：合肥工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O629.73

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