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Directed Evolution of Orthogonal Pyrrolysyl-tRNA Synthetases in Escherichia coli for the Genetic Encoding of Noncanonical Amino Acids

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Noncanonical Amino Acids

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1728))

Abstract

The directed evolution of orthogonal aminoacyl-tRNA synthetases (aaRS) for the genetic encoding of noncanonical amino acids (ncAA) has paved the way for the site-specific incorporation of >170 functionally diverse ncAAs into proteins in a large number of organisms [1, 2]. Here, we describe the directed evolution of orthogonal pyrrolysyl-tRNA synthetase (PylRS) mutants with new amino acid selectivities from libraries using a two-step selection protocol based on chloramphenicol and barnase reporter systems. Although this protocol focuses on the evolution of PylRS variants, this procedure can be universally employed to evolve orthogonal aaRS.

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Acknowledgments

We acknowledge support by the TU Dortmund, the University of Konstanz and the Konstanz Research School Chemical Biology. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SU-726/2-2 in SPP1623 and SU-726/4-2 in SPP1601).

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Authors and Affiliations

  1. University of Konstanz, Constance, Germany

    Moritz J. Schmidt & Daniel Summerer

  2. Department of Chemistry and Chemical Biology, TU Dortmund University, Dortmund, Germany

    Daniel Summerer

Authors
  1. Moritz J. Schmidt
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  2. Daniel Summerer
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Corresponding author

Correspondence to Daniel Summerer .

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Editors and Affiliations

  1. Structural and Computational Biology Unit & Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany

    Edward A. Lemke

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Schmidt, M.J., Summerer, D. (2018). Directed Evolution of Orthogonal Pyrrolysyl-tRNA Synthetases in Escherichia coli for the Genetic Encoding of Noncanonical Amino Acids. In: Lemke, E. (eds) Noncanonical Amino Acids. Methods in Molecular Biology, vol 1728. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7574-7_5

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  • DOI: https://doi.org/10.1007/978-1-4939-7574-7_5

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7573-0

  • Online ISBN: 978-1-4939-7574-7

  • eBook Packages: Springer Protocols

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