定向改造酵母释放因子eRF1识别特异性的研究

对蛋白质进行定点插入非传统氨基酸的改造是研究蛋白质结构、功能和复合体变化的重要工具，也可为药物和疫苗的研发提供新策略。现有研究大多利用琥珀密码子（UAG）编码非传统氨基酸，而真核生物释放因子eRF1也可以识别UAG密码子并终止蛋白质的翻译，导致无法高效率合成含有定点插入非传统氨基酸的蛋白质。本课题组参与的酿酒酵母基因组合成计划拟将其基因组内所有的UAG终止密码子替换为UAA终止密码子，这将为在酵母中利用UAG密码子进行定点插入非传统氨基酸的蛋白质改造奠定基础。本项目将开展酿酒酵母释放因子eRF1的改造研究，结合理性设计和蛋白质定向进化技术改变eRF1识别终止密码子的特异性，并消除其识别UAG终止密码子的能力，最终突破合成酵母内含有定点插入非传统氨基酸蛋白质合成的技术瓶颈。本项目的完成将有助于揭示真核生物eRF1特异性识别终止密码子的分子机制，并为合成酵母成功的应用于蛋白质改造奠定基础。

Expansion of the genetic code allows unnatural amino acids to be site-specifically incorporated into protein in live biological systems, which is contributing and transforming the study, manipulation, and evolution of proteins by expanding the set of building blocks in protein with new chemical and physical properties. However, the efficiency of unnatural amino acid incorporation in response to the amber stop codon (UAG) in yeast cells is commonly considered to be low. In the synthetic yeast genome project (Sc2.0), one of the design principles was to eliminate the UAG stop codon by recoding to UAA, in order to free the UAG codon for the site-specific introduction of unnatural amino acids into protein in yeast cells. In this project, we plan to rationally engineer eRF1, which normally terminates translation at all tree stop codon (UAG, UAA, and UGA), to provide a substantial increase in unnatural amino acid incorporation by eliminating the cognition of UAG codon without increasing readthrough of other stop codons. This study could remove the technological bottleneck of the site-specific introduction of unnatural amino acids into proteins in yeast cells, and contribute to understanding the mechanisms of specific recognition of three stop codons by release factor eRF1.