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

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

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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, pp. 97-111. ISBN 978-1-4939-7573-0. Available under: doi: 10.1007/978-1-4939-7574-7_5

@incollection{Schmidt2018Direc-42824, title={Directed Evolution of Orthogonal Pyrrolysyl-tRNA Synthetases in Escherichia coli for the Genetic Encoding of Noncanonical Amino Acids}, year={2018}, doi={10.1007/978-1-4939-7574-7_5}, number={1728}, isbn={978-1-4939-7573-0}, address={New York, NY}, publisher={Humana Press}, 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} }

Schmidt, Moritz J. 2018-07-09T12:43:16Z Schmidt, Moritz J. 2018 Directed Evolution of Orthogonal Pyrrolysyl-tRNA Synthetases in Escherichia coli for the Genetic Encoding of Noncanonical Amino Acids eng Summerer, Daniel Summerer, Daniel 2018-07-09T12:43:16Z 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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