title: Performance analysis of orthogonal pairs designed for an expanded eukaryotic genetic code.
abstract: BACKGROUND
  The suppression of amber stop codons with non-canonical amino acids (ncAAs) is used for the site-specific introduction of many unusual functions into proteins. Specific orthogonal aminoacyl-tRNA synthetase (o-aaRS)/amber suppressor tRNA(CUA) pairs (o-pairs) for the incorporation of ncAAs in S. cerevisiae were previously selected from an E. coli tyrosyl-tRNA synthetase/tRNA(CUA) mutant library. Incorporation fidelity relies on the specificity of the o-aaRSs for their ncAAs and the ability to effectively discriminate against their natural substrate Tyr or any other canonical amino acid.
  
  
  RESULTS
  We used o-pairs previously developed for ncAAs carrying reactive alkyne-, azido-, or photocrosslinker side chains to suppress an amber mutant of human superoxide dismutase 1 in S. cerevisiae. We found worse incorporation efficiencies of the alkyne- and the photocrosslinker ncAAs than reported earlier. In our hands, amber suppression with the ncAA containing the azido group did not occur at all. In addition to the incorporation experiments in S. cerevisiae, we analyzed the catalytic properties of the o-aaRSs in vitro. Surprisingly, all o-aaRSs showed much higher preference for their natural substrate Tyr than for any of the tested ncAAs. While it is unclear why efficiently recognized Tyr is not inserted at amber codons, we speculate that metabolically inert ncAAs accumulate in the cell, and for this reason they are incorporated despite being weak substrates for the o-aaRSs.
  
  
  CONCLUSIONS
  O-pairs have been developed for a whole plethora of ncAAs. However, a systematic and detailed analysis of their catalytic properties is still missing. Our study provides a comprehensive scrutiny of o-pairs developed for the site-specific incorporation of reactive ncAAs in S. cerevisiae. It suggests that future development of o-pairs as efficient biotechnological tools will greatly benefit from sound characterization in vivo and in vitro in parallel to monitoring intracellular ncAA levels.
authors.affiliation: Department of Biocatalysis, Technical University of Berlin, Berlin, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Nehring
authors.forename: Nediljko
authors.initials: N
authors.lastname: Budisa
authors.forename: Birgit
authors.initials: B
authors.lastname: Wiltschi
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018388:Codon, Terminator / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli; D056890:Eukaryota; D005815:Genetic Code; D005818:Genetic Engineering; D005822:Genetic Vectors; D015698:Genomic Library; D006801:Humans; D009154:Mutation; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000072105:Superoxide Dismutase-1; D014443:Tyrosine / Q000031:analogs & derivatives / Q000235:genetics*
chemical_list: D018388:Codon, Terminator; C000606290:SOD1 protein, human; D029701:Saccharomyces cerevisiae Proteins; D014443:Tyrosine; D012343:RNA, Transfer; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D000604:Amino Acyl-tRNA Synthetases