Reprogramming Initiator and Nonsense Codons to Simultaneously Install Three Distinct Noncanonical Amino Acids into Proteins in E. coli

Methods Mol Biol. 2023:2676:101-116. doi: 10.1007/978-1-0716-3251-2_7.

Abstract

Multiple noncanonical amino acids can be installed into proteins in E. coli using mutually orthogonal aminoacyl-tRNA synthetase and tRNA pairs. Here we describe a protocol for simultaneously installing three distinct noncanonical amino acids into proteins for site-specific bioconjugation at three sites. This method relies on an engineered, UAU-suppressing, initiator tRNA, which is aminoacylated with a noncanonical amino acid by Methanocaldococcus jannaschii tyrosyl-tRNA synthetase. Using this initiator tRNA/aminoacyl-tRNA synthetase pair, together with the pyrrolysyl-tRNA synthetase/tRNAPyl pairs from Methanosarcina mazei and Ca. Methanomethylophilus alvus, three noncanonical amino acids can be installed into proteins in response to the UAU, UAG, and UAA codons.

Keywords: Bioorthogonal chemistry; Genetic code expansion; Non-canonical amino acids; Pyrrolysyl-tRNA synthetase; Synthetic biology.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acids* / chemistry
  • Amino Acyl-tRNA Synthetases* / metabolism
  • Codon, Nonsense
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Proteins / metabolism
  • RNA, Transfer / metabolism
  • RNA, Transfer, Met / metabolism

Substances

  • Amino Acids
  • Codon, Nonsense
  • RNA, Transfer, Met
  • Proteins
  • RNA, Transfer
  • Amino Acyl-tRNA Synthetases