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Protein Expression with Biosynthesized Noncanonical Amino Acids

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Genetically Incorporated Non-Canonical Amino Acids

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2676))

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Abstract

Natural proteins are normally made by 20 canonical amino acids. Genetic code expansion (GCE) enables incorporation of diverse chemically synthesized noncanonical amino acids (ncAAs) by orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs using nonsense codons, which could significantly expand new functionalities of proteins in both scientific and biomedical applications. Here, by hijacking the cysteine biosynthetic enzymes, we describe a method combining amino acid biosynthesis and GCE to introduce around 50 structurally novel ncAAs into proteins by supplementation of commercially available aromatic thiol precursors, thus eliminating the need to chemically synthesize these ncAAs. A screening method is also provided for improving the incorporation efficiency of a particular ncAA. Furthermore, we demonstrate bioorthogonal groups, such as azide and ketone, that are compatible with our system and can be easily introduced into protein for subsequent site-specific labeling.

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Author notes

  1. Authors Yong Wang and Wenkang Cai have contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Natural and Biomimetic Drugs, Chemical Biology Center, Department of Molecular and Cellular Pharmacology, Pharmaceutical Sciences, Peking University, Beijing, China

    Yong Wang, Wenkang Cai, Boyang Han & Tao Liu

Authors
  1. Yong Wang
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  2. Wenkang Cai
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  3. Boyang Han
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  4. Tao Liu
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Corresponding author

Correspondence to Tao Liu .

Editor information

Editors and Affiliations

  1. Institute of Molecular Physiology, Shenzhen Bay Laboratory, Shenzhen, China

    Yu-Hsuan Tsai

  2. Laboratory of Synthetic and Systems Biology, Science for Life Laboratory, Department of Medical Biochemistry and Biophysics, Division of Genome Biology, Karolinska Institutet, Solna, Stockholm, Sweden

    Simon J. Elsässer

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Wang, Y., Cai, W., Han, B., Liu, T. (2023). Protein Expression with Biosynthesized Noncanonical Amino Acids. In: Tsai, YH., Elsässer, S.J. (eds) Genetically Incorporated Non-Canonical Amino Acids. Methods in Molecular Biology, vol 2676. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3251-2_6

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  • DOI: https://doi.org/10.1007/978-1-0716-3251-2_6

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3250-5

  • Online ISBN: 978-1-0716-3251-2

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