Wu Yang, Wang Zhaoguan, Qiao Xin, Li Jiaojiao, Shu Xiangrong, Qi Hao
School of Chemical Engineering and Technology, Tianjin University, Tianjin, China.
Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin, China.
Front Bioeng Biotechnol. 2020 Jul 22;8:863. doi: 10.3389/fbioe.2020.00863. eCollection 2020.
Cell-free protein synthesis (CFPS) has emerged as a novel protein expression platform. Especially the incorporation of non-canonical amino acids (ncAAs) has led to the development of numerous flexible methods for efficient and extensive expression of artificial proteins. Approaches were developed to eliminate the endogenous competition for ncAAs and engineer translation factors, which significantly enhanced the incorporation efficiency. Furthermore, aminoacylation methods can be conveniently combined with cell-free systems, extensively expanding the available ncAAs with novel and unique moieties. In this review, we summarize the recent progresses on the efficient and extensive incorporation of ncAAs by different strategies based on the elimination of competition by endogenous factors, translation factors engineering and extensive incorporation of novel ncAAs coupled with aminoacylation methods in CFPS. We also aim to offer new ideas to researchers working on ncAA incorporation techniques in CFPS and applications in various emerging fields.
无细胞蛋白质合成（CFPS）已成为一种新型蛋白质表达平台。特别是非天然氨基酸（ncAAs）的掺入，已催生出众多灵活方法，用于高效且广泛地表达人工蛋白质。人们开发了一些方法来消除对非天然氨基酸的内源性竞争并改造翻译因子，这显著提高了掺入效率。此外，氨酰化方法可方便地与无细胞系统相结合，用新颖独特的基团大幅扩展了可用的非天然氨基酸种类。在本综述中，我们总结了基于消除内源性因素竞争、翻译因子工程以及在无细胞蛋白质合成中结合氨酰化方法大量掺入新型非天然氨基酸等不同策略，在高效且广泛地掺入非天然氨基酸方面的最新进展。我们还旨在为从事无细胞蛋白质合成中非天然氨基酸掺入技术及在各个新兴领域应用研究的人员提供新思路。
