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Methods Mol Biol. 2018;1728:97-111. doi: 10.1007/978-1-4939-7574-7_5.

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

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


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.


Amber suppression; Directed evolution; Expanded genetic code; Genetic selection; Noncanonical amino acids; Pyrrolysyl-tRNA synthetase

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