Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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


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.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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

Accession codes



Data deposits

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


  1. Tsien, R.Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).

    CAS  Article  Google Scholar 

  2. Zhang, J., Campbell, R.E., Ting, A.Y. & Tsien, R.Y. Creating new fluorescent probes for cell biology. Nat. Rev. Mol. Cell Biol. 3, 906–918 (2002).

    CAS  Article  Google Scholar 

  3. Lauf, U., Lopez, P. & Falk, M.M. Expression of fluorescently tagged connexins: a novel approach to rescue function of oligomeric DsRed-tagged proteins. FEBS Lett. 498, 11–15 (2001).

    CAS  Article  Google Scholar 

  4. Matz, M.V. et al. Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol. 17, 969–973 (1999).

    CAS  Article  Google Scholar 

  5. Baird, G.S., Zacharias, D.A. & Tsien, R.Y. Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. USA 97, 11984–11989 (2000).

    CAS  Article  Google Scholar 

  6. Bevis, B.J. & Glick, B.S. Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed). Nat. Biotechnol. 20, 83–87 (2002).

    CAS  Article  Google Scholar 

  7. Campbell, R.E. et al. A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. USA 99, 7877–7882 (2002).

    CAS  Article  Google Scholar 

  8. Gross, L.A., Baird, G.S., Hoffman, R.C., Baldridge, K.K. & Tsien, R.Y. The structure of the chromophore within DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. USA 97, 11990–11995 (2000).

    CAS  Article  Google Scholar 

  9. Rusan, N.M., Fagerstrom, C.J., Yvon, A.M. & Wadsworth, P. Cell cycle-dependent changes in microtubule dynamics in living cells expressing green fluorescent protein-alpha tubulin. Mol. Biol. Cell 12, 971–980 (2001).

    CAS  Article  Google Scholar 

  10. Verkhusha, V.V. & Lukyanov, K.A. The molecular properties and applications of Anthozoa fluorescent proteins and chromoproteins. Nat. Biotechnol. 22, 289–296 (2004).

    CAS  Article  Google Scholar 

  11. Heim, R., Cubitt, A.B. & Tsien, R.Y. Improved green fluorescence. Nature 373, 663–664 (1995).

    CAS  Article  Google Scholar 

  12. Heim, R., Prasher, D.C. & Tsien, R.Y. Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc. Natl. Acad. Sci. USA 91, 12501–12504 (1994).

    CAS  Article  Google Scholar 

  13. Ip, D.T. et al. Crystallization and preliminary crystallographic analysis of a novel orange fluorescent protein from the Cnidaria tube anemone Cerianthus sp. Acta Crystallogr. D Biol. Crystallogr. 60, 340–341 (2004).

    Article  Google Scholar 

  14. Karasawa, S., Araki, T., Nagai, T., Mizuno, H. & Miyawaki, A. Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer. Biochem. J. 381, 307–312 (2004).

    CAS  Article  Google Scholar 

  15. Llopis, J., McCaffery, J.M., Miyawaki, A., Farquhar, M.G. & Tsien, R.Y. Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins. Proc. Natl. Acad. Sci. USA 95, 6803–6808 (1998).

    CAS  Article  Google Scholar 

  16. Zapata-Hommer, O. & Griesbeck, O. Efficiently folding and circularly permuted variants of the Sapphire mutant of GFP. BMC Biotechnol. 3, 5 (2003). (

    Article  Google Scholar 

  17. Griesbeck, O., Baird, G.S., Campbell, R.E., Zacharias, D.A. & Tsien, R.Y. Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J. Biol. Chem. 276, 29188–29194 (2001).

    CAS  Article  Google Scholar 

  18. Miyawaki, A. & Tsien, R.Y. Monitoring protein conformations and interactions by fluorescence resonance energy transfer between mutants of green fluorescent protein. Methods Enzymol. 327, 472–500 (2000).

    CAS  Article  Google Scholar 

  19. Hadjantonakis, A.K., Dickinson, M.E., Fraser, S.E. & Papaioannou, V.E. Technicolour transgenics: imaging tools for functional genomics in the mouse. Nat. Rev. Genet. 4, 613–625 (2003).

    CAS  Article  Google Scholar 

  20. Farkas, D.L. et al. Non-invasive image acquisition and advanced processing in optical bioimaging. Comput. Med. Imaging Graph. 22, 89–102 (1998).

    CAS  Article  Google Scholar 

  21. Fradkov, A.F. et al. Far-red fluorescent tag for protein labelling. Biochem. J. 368, 17–21 (2002).

    CAS  Article  Google Scholar 

  22. Chudakov, D.M. et al. Kindling fluorescent proteins for precise in vivo photolabeling. Nat. Biotechnol. 21, 191–194 (2003).

    CAS  Article  Google Scholar 

  23. Mizuno, H. et al. Photo-induced peptide cleavage in the green-to-red conversion of a fluorescent protein. Mol. Cell 12, 1051–1058 (2003).

    CAS  Article  Google Scholar 

  24. Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H. & Miyawaki, A. An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc. Natl. Acad. Sci. USA 99, 12651–12656 (2002).

    CAS  Article  Google Scholar 

  25. Petersen, J. et al. The 2.0-A crystal structure of eqFP611, a far red fluorescent protein from the sea anemone Entacmaea quadricolor. J. Biol. Chem. 278, 44626–44631 (2003).

    CAS  Article  Google Scholar 

  26. Guzman, L.M., Belin, D., Carson, M.J. & Beckwith, J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177, 4121–4130 (1995).

    CAS  Article  Google Scholar 

  27. Baird, G.S., Zacharias, D.A. & Tsien, R.Y. Circular permutation and receptor insertion within green fluorescent proteins. Proc. Natl. Acad. Sci. USA 96, 11241–11246 (1999).

    CAS  Article  Google Scholar 

  28. Daugherty, P.S., Olsen, M.J., Iverson, B.L. & Georgiou, G. Development of an optimized expression system for the screening of antibody libraries displayed on the Escherichia coli surface. Protein Eng. 12, 613–621 (1999).

    CAS  Article  Google Scholar 

  29. Chiu, C.S., Kartalov, E., Unger, M., Quake, S. & Lester, H.A. Single-molecule measurements calibrate green fluorescent protein surface densities on transparent beads for use with 'knock-in' animals and other expression systems. J. Neurosci. Methods 105, 55–63 (2001).

    CAS  Article  Google Scholar 

  30. Ward, W.W. Biochemical and physical properties of GFP. in Green Fluorescent Protein: Properties, Applications, and Protocols (eds. Chalfie, M. & Kain, S.) 45–75 (Wiley, New York, 1998).

    Google Scholar 

Download references


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

Authors and Affiliations


Corresponding author

Correspondence to Roger Y Tsien.

Ethics declarations

Competing interests

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)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing