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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.
J Mol Biol. 2010 Nov 19; 404(1):70-87.JM

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+Show Affiliations

Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl für Biologische Chemie, Technische Universität München, 85350 Freising-Weihenstephan, Germany.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

20837025

Citation

Kuhn, Sebastian M., et al. "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." Journal of Molecular Biology, vol. 404, no. 1, 2010, pp. 70-87.
Kuhn SM, Rubini M, Fuhrmann M, et al. 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. J Mol Biol. 2010;404(1):70-87.
Kuhn, S. M., Rubini, M., Fuhrmann, M., Theobald, I., & Skerra, A. (2010). 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. Journal of Molecular Biology, 404(1), 70-87. https://doi.org/10.1016/j.jmb.2010.09.001
Kuhn SM, et al. 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. J Mol Biol. 2010 Nov 19;404(1):70-87. PubMed PMID: 20837025.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Kuhn,Sebastian M, AU - Rubini,Marina, AU - Fuhrmann,Markus, AU - Theobald,Ina, AU - Skerra,Arne, Y1 - 2010/09/15/ PY - 2010/05/24/received PY - 2010/08/14/revised PY - 2010/09/01/accepted PY - 2010/9/15/entrez PY - 2010/9/15/pubmed PY - 2010/12/14/medline SP - 70 EP - 87 JF - Journal of molecular biology JO - J. Mol. Biol. VL - 404 IS - 1 N2 - 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. SN - 1089-8638 UR - https://www.unboundmedicine.com/medline/citation/20837025/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_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0022-2836(10)00948-4 DB - PRIME DP - Unbound Medicine ER -