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在酵母表面构建含非标准氨基酸的蛋白质

Engineering Proteins Containing Noncanonical Amino Acids on the Yeast Surface.

作者信息

Hershman Rebecca L, Rezhdo Arlinda, Stieglitz Jessica T, Van Deventer James A

机构信息

Chemical and Biological Engineering Department, Tufts University, Medford, MA, USA.

Biomedical Engineering Department, Tufts University, Medford, MA, USA.

出版信息

Methods Mol Biol. 2022;2491:491-559. doi: 10.1007/978-1-0716-2285-8_23.

Abstract

Yeast display has been used to advance many critical research areas, including the discovery of unique protein binders and biological therapeutics. In parallel, noncanonical amino acids (ncAAs) have been used to tailor antibody-drug conjugates and enable discovery of therapeutic leads. Together, these two technologies have allowed for generation of synthetic antibody libraries, where the introduction of ncAAs in yeast-displayed proteins allows for library screening for therapeutically relevant targets. The combination of yeast display with genetically encoded ncAAs increases the available chemistry in proteins and advances applications that require high-throughput strategies. In this chapter, we discuss methods for displaying proteins containing ncAAs on the yeast surface, generating and screening libraries of proteins containing ncAAs, preparing bioconjugates on the yeast surface in large scale, generating and screening libraries of aminoacyl-tRNA synthetases (aaRSs) for encoding ncAAs by using reporter constructs, and characterizing ncAA-containing proteins secreted from yeast. The experimental designs laid out in this chapter are generalizable for discovery of protein binders to a variety of targets and aaRS evolution to continue expanding the genetic code beyond what is currently available in yeast.

摘要

酵母展示技术已被用于推动许多关键研究领域的发展,包括发现独特的蛋白质结合物和生物治疗药物。与此同时,非天然氨基酸(ncAAs)已被用于定制抗体-药物偶联物,并有助于发现治疗先导物。这两种技术共同促成了合成抗体文库的产生,其中在酵母展示的蛋白质中引入非天然氨基酸可用于筛选与治疗相关的靶点的文库。酵母展示与基因编码的非天然氨基酸相结合,增加了蛋白质中可用的化学性质,并推动了需要高通量策略的应用。在本章中,我们将讨论在酵母表面展示含有非天然氨基酸的蛋白质的方法、生成和筛选含有非天然氨基酸的蛋白质文库、大规模制备酵母表面的生物偶联物、使用报告构建体生成和筛选用于编码非天然氨基酸的氨酰-tRNA合成酶(aaRSs)文库,以及表征酵母分泌的含非天然氨基酸的蛋白质。本章中列出的实验设计可普遍应用于发现针对各种靶点的蛋白质结合物,以及进行氨酰-tRNA合成酶进化,以继续扩展酵母目前可用的遗传密码。

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