title: 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.
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 eGFP(UAG) 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.
authors.affiliation: Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl für Biologische Chemie, Technische Universität München, 85350 Freising-Weihenstephan, Germany.
authors.forename: Sebastian M
authors.initials: SM
authors.lastname: Kuhn
authors.forename: Marina
authors.initials: M
authors.lastname: Rubini
authors.forename: Markus
authors.initials: M
authors.lastname: Fuhrmann
authors.forename: Ina
authors.initials: I
authors.lastname: Theobald
authors.forename: Arne
authors.initials: A
authors.lastname: Skerra
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics* / Q000378:metabolism*; D018389:Codon, Nonsense; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008781:Methyltyrosines / Q000378:metabolism*; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D013489:Suppression, Genetic
chemical_list: D018389:Codon, Nonsense; D008781:Methyltyrosines; D050505:Mutant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C002350:O-methyltyrosine; D000604:Amino Acyl-tRNA Synthetases