An enhanced system for unnatural amino acid mutagenesis in E. coli

J Mol Biol. 2010 Jan 15;395(2):361-74. doi: 10.1016/j.jmb.2009.10.030. Epub 2009 Oct 21.


We report a new vector, pEVOL, for the incorporation of unnatural amino acids into proteins in Escherichia coli using evolved Methanocaldococcus jannaschii aminoacyl-tRNA synthetase(s) (aaRS)/suppressor tRNA pairs. This new system affords higher yields of mutant proteins through the use of both constitutive and inducible promoters to drive the transcription of two copies of the M. jannaschii aaRS gene. Yields were further increased by coupling the dual-aaRS promoter system with a newly optimized suppressor tRNA(CUA)(opt) in a single-vector construct. The optimized suppressor tRNA(CUA)(opt) afforded increased plasmid stability compared with previously reported vectors for unnatural amino acid mutagenesis. To demonstrate the utility of this new system, we introduced 14 mutant aaRS into pEVOL and compared their ability to insert unnatural amino acids in response to three independent amber nonsense codons in sperm whale myoglobin or green fluorescent protein. When cultured in rich media in shake flasks, pEVOL was capable of producing more than 100 mg/L mutant GroEL protein. The versatility, increased yields, and increased stability of the pEVOL vector will further facilitate the expression of proteins with unnatural amino acids.

Publication types

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

MeSH terms

  • Amino Acids / chemistry
  • Amino Acids / genetics*
  • Amino Acids / metabolism*
  • Amino Acyl-tRNA Synthetases / genetics
  • Amino Acyl-tRNA Synthetases / metabolism
  • Base Sequence
  • Chaperonin 60 / genetics
  • Directed Molecular Evolution
  • Escherichia coli / genetics*
  • Escherichia coli / growth & development
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Gene Expression
  • Genes, Archaeal
  • Genetic Vectors*
  • Methanococcales / enzymology
  • Methanococcales / genetics
  • Molecular Sequence Data
  • Mutagenesis
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism
  • Nucleic Acid Conformation
  • Plasmids / genetics
  • RNA, Transfer / chemistry
  • RNA, Transfer / genetics


  • Amino Acids
  • Chaperonin 60
  • Escherichia coli Proteins
  • Mutant Proteins
  • RNA, Transfer
  • Amino Acyl-tRNA Synthetases