One plasmid selection system for the rapid evolution of aminoacyl-tRNA synthetases

Dual positive/negative selection vectors which allow the facile evolution of aminoacyl-tRNA synthetases are reported. We have developed a rapid, straightforward, one plasmid dual positive/negative selection system for the evolution of aminoacyl-tRNA synthetases with altered specificities in Escheric...

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Veröffentlicht in:Bioorganic & medicinal chemistry letters 2009-07, Vol.19 (14), p.3845-3847
Hauptverfasser: Melançon, Charles E., Schultz, Peter G.
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Sprache:eng
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Zusammenfassung:Dual positive/negative selection vectors which allow the facile evolution of aminoacyl-tRNA synthetases are reported. We have developed a rapid, straightforward, one plasmid dual positive/negative selection system for the evolution of aminoacyl-tRNA synthetases with altered specificities in Escherichia coli. This system utilizes an amber stop codon containing chloramphenicol acetyltransferase/uracil phosphoribosyltransferase fusion gene. We demonstrate the utility of the system by identifying a variant of the Methanococcus jannaschii tyrosyl synthetase from a library of 10 9 variants that selectively incorporates para-iodophenylalanine in response to an amber stop codon.
ISSN:0960-894X
1464-3405
DOI:10.1016/j.bmcl.2009.04.007