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Focused Engineering of Pyrrolysyl-tRNA Synthetase-Based Orthogonal Translation Systems for the Incorporation of Various 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))

Abstract

The expansion of the genetic code has become a valuable tool for molecular biology, biochemistry, and biotechnology. The pyrrolysyl-tRNA synthetase (PylRS) variants with their cognate tRNAPyl derived from methanogenic archaea of the genus Methanosarcina are the most popular tools for ribosomally mediated site-specific and proteome-wide statistical incorporation of noncanonical amino acids (ncAAs) into proteins. The incorporation of ncAAs can be used for numerous biotechnological and even therapeutically relevant applications. Here we present a protocol of engineering PylRS for novel substrates with unique chemical functionalities. These functional groups can act as intrinsic probes, especially in complex biological environments such as mammalian cells, tissues, and even whole animals.

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Author information

Authors and Affiliations

  1. Bioanalytics Group, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany

    Nikolaj G. Koch

  2. Biocatalysis Group, Institute of Chemistry, Technische Universität Berlin, Berlin, Germany

    Nikolaj G. Koch & Nediljko Budisa

  3. Chemical Synthetic Biology, Department of Chemistry, University of Manitoba, Winnipeg, MB, Canada

    Nediljko Budisa

Authors
  1. Nikolaj G. Koch
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  2. Nediljko Budisa
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Corresponding author

Correspondence to Nediljko Budisa .

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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Koch, N.G., Budisa, N. (2023). Focused Engineering of Pyrrolysyl-tRNA Synthetase-Based Orthogonal Translation Systems for the Incorporation of Various 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_1

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  • DOI: https://doi.org/10.1007/978-1-0716-3251-2_1

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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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