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

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


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.

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

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids / chemistry
  • Amino Acids / genetics*
  • Amino Acyl-tRNA Synthetases / genetics*
  • Amino Acyl-tRNA Synthetases / metabolism
  • Codon*
  • Culture Media
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism*
  • Evolution, Molecular*
  • Fluorescent Antibody Technique
  • Gene Expression
  • Gene Order
  • Lysine / analogs & derivatives*
  • Lysine / metabolism
  • Plasmids / genetics


  • Amino Acids
  • Codon
  • Culture Media
  • Amino Acyl-tRNA Synthetases
  • pyrrolysine
  • Lysine