用于跨领域编码非天然氨基酸的正交 tRNA/合成酶对的选择和验证。
Selection and validation of orthogonal tRNA/synthetase pairs for the encoding of unnatural amino acids across kingdoms.
机构信息
Department of Molecular Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA, United States; Unnatural Protein Facility, Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, United States.
Department of Molecular Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA, United States.
出版信息
Methods Enzymol. 2021;654:3-18. doi: 10.1016/bs.mie.2021.03.009. Epub 2021 Apr 9.
As an increasing number of protein structures are resolved at atomic and near-atomic resolution, conventional amino acid mutagenesis may be insufficient to test many mechanistic hypotheses. As a result, the development of new tRNA/aminoacyl-tRNA synthetase (aaRS) pairs has become an important tool for determining intricate molecular interactions and understanding protein structures. This chapter describes in detail the directed evolution of new tRNA/aaRS pairs in Escherichia coli for the incorporation of non-canonical amino acids (ncAA). Section 1 describes the selection of new tRNA/aaRS pairs in E. coli. Section 2 details the use of a synthetase to incorporate an ncAA into a mammalian cell line, and Sections 1 and 2 both include methods on the determination of synthetase efficacy and fidelity.
随着越来越多的蛋白质结构以原子和近原子分辨率解析，传统的氨基酸诱变可能不足以测试许多机械假设。因此，开发新的 tRNA/氨酰-tRNA 合成酶（aaRS）对成为确定复杂分子相互作用和理解蛋白质结构的重要工具。本章详细描述了大肠杆菌中新 tRNA/aaRS 对的定向进化，以掺入非典型氨基酸（ncAA）。第 1 节描述了在大肠杆菌中选择新的 tRNA/aaRS 对。第 2 节详细介绍了使用合成酶将 ncAA 掺入哺乳动物细胞系的方法，第 1 节和第 2 节都包括关于测定合成酶功效和保真度的方法。
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