Engineering of an orthogonal aminoacyl-tRNA synthetase for efficient incorporation of the non-natural amino acid O-methyl-L-tyrosine using fluorescence-based bacterial cell sorting

Sebastian M. Kuhn, Marina Rubini, Markus Fuhrmann, Ina Theobald, Arne Skerra

Research output: Contribution to journalArticlepeer-review

31 Scopus citations

Abstract

We describe a strategy for the rapid selection of mutant aminoacyl-tRNA synthetases (aaRS) with specificity for a novel amino acid based on fluorescence-activated cell sorting of transformed Escherichia coli using as reporter the enhanced green fluorescent protein (eGFP) whose gene carries an amber stop codon (TAG) at a permissive site upstream of the fluorophore. To this end, a one-plasmid expression system was developed encoding an inducible modified Methanocaldococcus jannaschii (Mj) tyrosyl-tRNA synthetase, the orthogonal cognate suppressor tRNA, and eGFPUAG in an individually regulatable fashion. Using this system a previously described aaRS with specificity for O-methyl-L-tyrosine (MeTyr) was engineered for 10-fold improved incorporation of the foreign amino acid by selection from a mutant library, prepared by error-prone as well as focused random mutagenesis, for MeTyr-dependent eGFP fluorescence. Applying alternating cycles of positive and negative fluorescence-activated bacterial cell sorting in the presence or in the absence, respectively, of the foreign amino acid was crucial to select for high specificity of MeTyr incorporation. The optimized synthetase was used for the preparative expression of a modified uvGFP carrying MeTyr at position 66 as part of its fluorophore. This biosynthetic protein showed quantitative incorporation of the non-natural amino acid, as determined by mass spectrometry, and it revealed a unique emission spectrum due to the altered chemical structure of its fluorophore. Our combined genetic/selection system offers advantages over earlier approaches that relied wholly or in part on antibiotic selection schemes, and it should be generally useful for the engineering and optimization of orthogonal aaRS/tRNA pairs to incorporate non-natural amino acids into recombinant proteins.

Original languageEnglish
Pages (from-to)70-87
Number of pages18
JournalJournal of Molecular Biology
Volume404
Issue number1
DOIs
StatePublished - 19 Nov 2010

Keywords

  • E. coli expression
  • Fluorescence-activated cell sorting
  • Genetic code
  • Green fluorescent protein
  • Non-canonical amino acid

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