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

Schmidt, Moritz J.; Summerer, Daniel

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

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Datum

2018

Publikationstyp

Beitrag zu einem Sammelband

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Published

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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

Zusammenfassung

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.

Fachgebiet (DDC)

540 Chemie

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ISO 690

SCHMIDT, 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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