Book chapter
Selection and validation of orthogonal tRNA/synthetase pairs for the encoding of unnatural amino acids across kingdoms
Ion Channels: Channel Chemical Biology, Engineering, and Physiological Function, Vol.654, pp.3-18
Methods in enzymology, 654, Elsevier
01/01/2021
DOI: 10.1016/bs.mie.2021.03.009
Abstract
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 1 Introduction, 2 Screening of the pyrrolysine synthetase library in both include methods on the determination of synthetase efficacy and fidelity.
Details
- Title: Subtitle
- Selection and validation of orthogonal tRNA/synthetase pairs for the encoding of unnatural amino acids across kingdoms
- Creators
- Grace D Galles - University of Iowa, Molecular Physiology and BiophysicsDaniel T Infield - University of Iowa, Molecular Physiology and BiophysicsRyan A Mehl - Unnatural Protein Facility, Department of Biochemistry and Biophysics, Oregon State UniversityChristopher A Ahern - University of Iowa, Molecular Physiology and Biophysics
- Resource Type
- Book chapter
- Publication Details
- Ion Channels: Channel Chemical Biology, Engineering, and Physiological Function, Vol.654, pp.3-18
- Series
- Methods in enzymology; 654
- DOI
- 10.1016/bs.mie.2021.03.009
- ISSN
- 0076-6879
- eISSN
- 1557-7988
- Publisher
- Elsevier
- Grant note
- DOI: 10.13039/100000002, name: National Institutes of Health, award: R01GM131168, F32HL149184, R24NS104617
- Language
- English
- Date published
- 01/01/2021
- Academic Unit
- Molecular Physiology and Biophysics; Iowa Neuroscience Institute
- Record Identifier
- 9984215756502771
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