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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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2018

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https://doi.org/10.1007/978-1-4939-7574-7_5
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LEMKE, Edward A., ed.. Noncanonical amino acids : methods and protocols. New York, NY: Humana Press, 2018, pp. 97-111. Methods in molecular biology. 1728. ISBN 978-1-4939-7573-0. Available under: doi: 10.1007/978-1-4939-7574-7_5

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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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ISO 690SCHMIDT, Moritz J., Daniel SUMMERER, 2018. Directed Evolution of Orthogonal Pyrrolysyl-tRNA Synthetases in Escherichia coli for the Genetic Encoding of Noncanonical Amino Acids. In: LEMKE, Edward A., ed.. Noncanonical amino acids : methods and protocols. New York, NY: Humana Press, 2018, pp. 97-111. Methods in molecular biology. 1728. ISBN 978-1-4939-7573-0. Available under: doi: 10.1007/978-1-4939-7574-7_5
BibTex
@incollection{Schmidt2018Direc-42824,
  year={2018},
  doi={10.1007/978-1-4939-7574-7_5},
  title={Directed Evolution of Orthogonal Pyrrolysyl-tRNA Synthetases in Escherichia coli for the Genetic Encoding of Noncanonical Amino Acids},
  number={1728},
  isbn={978-1-4939-7573-0},
  publisher={Humana Press},
  address={New York, NY},
  series={Methods in molecular biology},
  booktitle={Noncanonical amino acids : methods and protocols},
  pages={97--111},
  editor={Lemke, Edward A.},
  author={Schmidt, Moritz J. and Summerer, Daniel}
}
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