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Reprogramming Initiator and Nonsense Codons to Simultaneously Install Three Distinct Noncanonical Amino Acids into Proteins in E. coli

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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

Multiple noncanonical amino acids can be installed into proteins in E. coli using mutually orthogonal aminoacyl-tRNA synthetase and tRNA pairs. Here we describe a protocol for simultaneously installing three distinct noncanonical amino acids into proteins for site-specific bioconjugation at three sites. This method relies on an engineered, UAU-suppressing, initiator tRNA, which is aminoacylated with a noncanonical amino acid by Methanocaldococcus jannaschii tyrosyl-tRNA synthetase. Using this initiator tRNA/aminoacyl-tRNA synthetase pair, together with the pyrrolysyl-tRNA synthetase/tRNAPyl pairs from Methanosarcina mazei and Ca. Methanomethylophilus alvus, three noncanonical amino acids can be installed into proteins in response to the UAU, UAG, and UAA codons.

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Acknowledgments

The methods described in this protocol were developed in collaboration with the Center for Genetically Encoded Materials, an NSF Center for Chemical Innovation (CHE-2002182). The authors thank Professors Alanna Schepartz and Dieter Söll for valuable advice on preparing the manuscript. Han-Kai Jiang holds a graduate student fellowship from the Taiwan Academic Talents Overseas Advancement Program from the Ministry of Science and Technology (MOST 110-2917-I-007-006). Jeffery M. Tharp is supported by a Pathway to Independence Award from the National Institute of General Medical Sciences of the National Institutes of Health under award number K99GM141320. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Authors and Affiliations

  1. Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT, USA

    Han-Kai Jiang

  2. Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan

    Han-Kai Jiang

  3. Chemical Biology & Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan

    Han-Kai Jiang

  4. Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan

    Han-Kai Jiang

  5. Department of Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, USA

    Jeffery M. Tharp

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  1. Han-Kai Jiang
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  2. Jeffery M. Tharp
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Correspondence to Jeffery M. Tharp .

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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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Jiang, HK., Tharp, J.M. (2023). Reprogramming Initiator and Nonsense Codons to Simultaneously Install Three Distinct Noncanonical Amino Acids into Proteins in E. coli. 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_7

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  • DOI: https://doi.org/10.1007/978-1-0716-3251-2_7

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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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