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

被引:34
作者
Kuhn, Sebastian M. [1 ,2 ]
Rubini, Marina [1 ,2 ]
Fuhrmann, Markus [3 ]
Theobald, Ina [1 ,2 ]
Skerra, Arne [1 ,2 ]
机构
[1] Tech Univ Munich, Munich Ctr Integrated Prot Sci CIPS M, D-85350 Freising Weihenstephan, Germany
[2] Tech Univ Munich, Lehrstuhl Biol Chem, D-85350 Freising Weihenstephan, Germany
[3] Sloning BioTechnol GmbH, D-82178 Puchheim, Germany
关键词
E. coli expression; fluorescence-activated cell sorting; genetic code; green fluorescent protein; non-canonical amino acid; SITE-SPECIFIC INCORPORATION; GENETIC-CODE EXPANSION; IN-VIVO INCORPORATION; ESCHERICHIA-COLI; ANTIBODY FRAGMENT; PLASMID STABILITY; PROTEINS; MUTAGENESIS; EVOLUTION; SYSTEM;
D O I
10.1016/j.jmb.2010.09.001
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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. (C) 2010 Elsevier Ltd. All rights reserved.
引用
收藏
页码:70 / 87
页数:18
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