Incorporating, Quantifying, and Leveraging Noncanonical Amino Acids in Yeast.
Incorporating, Quantifying, and Leveraging Noncanonical Amino Acids in Yeast.
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DOI：
10.1007/978-1-0716-1811-0_21
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发表时间：
2022
影响因子：
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通讯作者：
J.T. Stieglitz;J. V. Van Deventer
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文献类型：
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作者：
J.T. Stieglitz;J. V. Van Deventer
遗传密码扩展在增强蛋白质化学多样性和功能方面取得了非凡的进步，但仍然迫切需要理解和设计遗传密码扩展系统以提高效率。在终止密码子处掺入非规范氨基酸 (ncAA) 提供了一种将独特化学引入蛋白质的位点特异性方法，但与野生型蛋白质相比，产量通常较低。用于 ncAA 掺入的强大平台支持化学多样性蛋白质的表达和评估，适用于广泛的应用。在酵母中，ncAA 已被用于研究动态细胞过程，例如蛋白质-蛋白质相互作用，并且还可以探索真核生物特异性生物学，例如表观遗传学。此外，酵母展示是一种用于高通量工程和筛选蛋白质特性的有利技术。本章介绍的协议描述了细胞内或酵母表面蛋白质中基于酵母的 ncAA 基因编码的详细方法。此外，还提出了使用生物正交化学反应修饰酵母表面蛋白质并评估反应效率的方法。最后，还包括用于制备涉及遗传密码扩展的文库的方案。可以针对许多生物和化学应用以高通量构建和筛选包含 ncAA 的蛋白质文库或编码 ncAA 所需的细胞机器文库。 ncAA 的有效掺入有助于阐明基础真核生物学，并改进酶和基因组工程工具，以进化能够更好地适应替代遗传密码的宿主细胞。
Genetic code expansion has allowed for extraordinary advances in enhancing protein chemical diversity and functionality, but there remains a critical need for understanding and engineering genetic code expansion systems for improved efficiency. Incorporation of noncanonical amino acids (ncAAs) at stop codons provides a site-specific method for introducing unique chemistry into proteins, though often at reduced yields compared to wild-type proteins. A powerful platform for ncAA incorporation supports both the expression and evaluation of chemically diverse proteins for a broad range of applications. In yeast, ncAAs have been used to study dynamic cellular processes such as protein–protein interactions and also allow for exploration of eukaryotic-specific biology such as epigenetics. Furthermore, yeast display is an advantageous technology for engineering and screening the properties of proteins in high throughput. The protocols presented in this chapter describe detailed methods for the yeast-based genetic encoding of ncAAs in proteins intracellularly or on the yeast surface. In addition, methods are presented for modifying proteins on the yeast surface using bioorthogonal chemical reactions and evaluating reaction efficiency. Finally, protocols are included for the preparation of libraries that involve genetic code expansion. Libraries of proteins that contain ncAAs or libraries of the cellular machinery required to encode ncAAs can be constructed and screened in high throughput for many biological and chemical applications. Efficient incorporation of ncAAs facilitates elucidation of fundamental eukaryotic biology and advances tools for enzyme and genome engineering to evolve host cells that are better able to accommodate alternative genetic codes.