Letter | Published:

Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein

Nature Biotechnology volume 22, pages 15671572 (2004) | Download Citation

Subjects

Abstract

Fluorescent proteins are genetically encoded, easily imaged reporters crucial in biology and biotechnology1,2. When a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function3. Unfortunately, all wild-type yellow-to-red fluorescent proteins reported so far are obligately tetrameric and often toxic or disruptive4,5. The first true monomer was mRFP1, derived from the Discosoma sp. fluorescent protein “DsRed” by directed evolution first to increase the speed of maturation6, then to break each subunit interface while restoring fluorescence, which cumulatively required 33 substitutions7. Although mRFP1 has already proven widely useful, several properties could bear improvement and more colors would be welcome. We report the next generation of monomers. The latest red version matures more completely, is more tolerant of N-terminal fusions and is over tenfold more photostable than mRFP1. Three monomers with distinguishable hues from yellow-orange to red-orange have higher quantum efficiencies.

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Accessions

GenBank/EMBL/DDBJ

Data deposits

All sequences have been deposited in GenBank, accession numbers AY678264 through AY678271.

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Acknowledgements

We thank Oliver Griesbeck for the kind donation of T-Sapphire, Coyt Jackson for FACS support, Brent Martin for α-tubulin cDNA, and Rene Meijer and Lei Wang for helpful discussion. Sequencing services were provided by the UCSD Cancer Center shared sequencing resource. N.C.S. is a Howard Hughes Medical Institute Predoctoral Fellow. Construction of tubulin fusions and mammalian cell imaging were conducted at the National Center for Microscopy and Imaging Research, which is supported by National Institutes of Health (NIH) grant RR04050 (to Mark H. Ellisman). This work was also supported by NIH and Department of Energy grants NS27177 and DE-FG-01ER63276.

Author information

Author notes

    • Robert E Campbell

    Present address: Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.

Affiliations

  1. Department of Pharmacology, University of California at San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA.

    • Nathan C Shaner
    • , Robert E Campbell
    • , Paul A Steinbach
    • , Amy E Palmer
    •  & Roger Y Tsien
  2. Department of Chemistry and Biochemistry, University of California at San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA.

    • Roger Y Tsien
  3. Department of Neurosciences, University of California at San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA.

    • Ben N G Giepmans
  4. National Center of Microscopy and Imaging Research, University of California at San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA.

    • Ben N G Giepmans
  5. Howard Hughes Medical Institute, University of California at San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA.

    • Roger Y Tsien

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Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Roger Y Tsien.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    T-Sapphire-mOrange FRET.

  2. 2.

    Supplementary Fig. 2

    Discrimination of E. coli transfected with six different fluorescent proteins.

  3. 3.

    Supplementary Fig. 3

    Photobleaching curves for new RFP variants.

  4. 4.

    Supplementary Notes

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DOI

https://doi.org/10.1038/nbt1037

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