Engineering Proteins Containing Noncanonical Amino Acids on the Yeast Surface.
Engineering Proteins Containing Noncanonical Amino Acids on the Yeast Surface.
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DOI：
10.1007/978-1-0716-2285-8_23
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发表时间：
2022-01-01
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影响因子：
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通讯作者：
Van Deventer, James A
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文献类型：
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作者：
Hershman, Rebecca L;Rezhdo, Arlinda;Van Deventer, James A
酵母展示已被用于推进许多关键的研究领域，包括发现独特的蛋白质结合物和生物治疗。与此同时，非规范氨基酸（ncAAs）已被用于定制抗体-药物偶联物，并能够发现治疗线索。总之，这两种技术已经允许生成合成抗体文库，其中在酵母显示的蛋白质中引入ncAAs允许文库筛选治疗相关靶点。酵母展示与基因编码ncaa的结合增加了蛋白质中的可用化学成分，并推进了需要高通量策略的应用。在本章中，我们讨论了在酵母表面展示含ncAAs蛋白的方法，含ncAAs蛋白文库的生成和筛选方法，在酵母表面大规模制备生物偶联物的方法，利用报告基因构建体生成和筛选编码ncAAs的氨基酰基trna合成酶（aars）文库的方法，以及酵母分泌的含ncaa蛋白的表征方法。本章中列出的实验设计可推广到各种靶点的蛋白质结合物的发现和aaRS的进化，以继续扩展目前在酵母中可用的遗传密码。
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 oftherapeutic 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 largescale, 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.