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

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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Figure 1: Excitation and emission spectra for new RFP variants.
Figure 2: Sequences and genealogy.
Figure 3: mCherry performance in fusion constructs.

Accession codes

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.

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Correspondence to Roger Y Tsien.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

T-Sapphire-mOrange FRET. (PDF 93 kb)

Supplementary Fig. 2

Discrimination of E. coli transfected with six different fluorescent proteins. (PDF 155 kb)

Supplementary Fig. 3

Photobleaching curves for new RFP variants. (PDF 188 kb)

Supplementary Notes (PDF 32 kb)

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Shaner, N., Campbell, R., Steinbach, P. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22, 1567–1572 (2004). https://doi.org/10.1038/nbt1037

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