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

Academic Article
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Overview

related to degree

  • Xiao, Han, Ph.D. in Chemical Biology, Scripps Research 2010 - 2015

authors

  • Chatterjee, A.
  • Xiao, Han
  • Schultz, Peter

publication date

  • September 2012

journal

  • Proceedings of the National Academy of Sciences of the United States of America  Journal

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.

subject areas

  • Amino Acids
  • Amino Acyl-tRNA Synthetases
  • Archaeoglobus fulgidus
  • Cloning, Molecular
  • Escherichia coli
  • Evolution, Molecular
  • Gene Library
  • Mutagenesis, Site-Directed
  • Plasmids
  • Protein Engineering
  • Pyrococcus horikoshii
  • RNA, Transfer
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Research

keywords

  • anticodon recognition
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Identity

PubMed Central ID

  • PMC3443146

International Standard Serial Number (ISSN)

  • 0027-8424

Digital Object Identifier (DOI)

  • 10.1073/pnas.1212454109

PubMed ID

  • 22927411
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Additional Document Info

start page

  • 14841

end page

  • 14846

volume

  • 109

issue

  • 37

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