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Proc Natl Acad Sci U S A. 2012 Sep 11;109(37):14841-6. doi: 10.1073/pnas.1212454109. Epub 2012 Aug 27.

Evolution of multiple, mutually orthogonal prolyl-tRNA synthetase/tRNA pairs for unnatural amino acid mutagenesis in Escherichia coli.

Author information

1
Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA.

Abstract

The site-specific incorporation of unnatural amino acids (UAAs) into proteins in living cells relies on an engineered tRNA/aminoacyl-tRNA synthetase (tRNA/aaRS) pair, orthogonal to the host cell, to deliver the UAA of choice in response to a unique nonsense or frameshift codon. Here we report the generation of mutually orthogonal prolyl-tRNA/prolyl-tRNA synthase (ProRS) pairs derived from an archaebacterial ancestor for use in Escherichia coli. By reprogramming the anticodon-binding pocket of Pyrococcus horikoshii ProRS (PhProRS), we were able to identify synthetase variants that recognize engineered Archaeoglobus fulgidus prolyl-tRNAs (Af-tRNA(Pro)) with three different anticodons: CUA, AGGG, and CUAG. Several of these evolved PhProRSs show specificity toward a particular anticodon variant of Af-tRNA(Pro), whereas others are promiscuous. Further evolution of the Af-tRNA(Pro) led to a variant exhibiting significantly improved amber suppression efficiency. Availability of a prolyl-tRNA/aaRS pair should enable site-specific incorporation of proline analogs and other N-modified UAAs into proteins in E. coli. The evolution of mutually orthogonal prolyl-tRNA/ProRS pairs demonstrates the plasticity of the tRNA-aaRS interface and should facilitate the incorporation of multiple, distinct UAAs into proteins.

PMID:
22927411
PMCID:
PMC3443146
DOI:
10.1073/pnas.1212454109
[Indexed for MEDLINE]
Free PMC Article

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