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Progress toward an expanded eukaryotic genetic code.

Jason W Chin1, T Ashton Cropp, Stephanie Chu

  • 1Department of Chemistry, Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

Chemistry & Biology
|July 3, 2003
PubMed
Summary
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Researchers developed a method to genetically engineer yeast to incorporate unnatural amino acids into proteins. This technique utilizes reporter genes and selection strategies to isolate specific aminoacyl-tRNA synthetase activities for advanced protein manipulation.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Synthetic Biology

Background:

  • Expanding the eukaryotic genetic code enables novel protein engineering.
  • Unnatural amino acids (UAAs) offer unique properties for protein function manipulation.
  • Efficient methods are needed to incorporate UAAs into proteins in eukaryotes.

Purpose of the Study:

  • To describe a general approach for isolating aminoacyl-tRNA synthetases (aaRS) that incorporate UAAs with high fidelity in Saccharomyces cerevisiae.
  • To enable the expansion of the eukaryotic genetic code for protein manipulation.

Main Methods:

  • Utilized GAL4-responsive reporter genes (HIS3, URA3, lacZ) activated by amber codon suppression.
  • Optimized GAL4 reporter gene constructs.
  • Demonstrated positive and negative selection of active Escherichia coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNA(CUA) pairs.

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  • Performed selections on a single cell with tunable stringency.

    • Main Results:

    • Successfully optimized GAL4 reporters for selection.
    • Demonstrated the efficacy of positive and negative selection for identifying functional aaRS/tRNA pairs.
    • Showcased the ability to perform dual selections within a single cell.

    Conclusions:

    • The described method facilitates the isolation of aaRS/tRNA(CUA) activities from large mutant libraries.
    • This approach is valuable for expanding the genetic code and engineering protein function in eukaryotes.
    • The method offers a versatile platform for discovering and optimizing UAA incorporation systems.