Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed)

  • A Corrigendum to this article was published on 01 November 2002


The red fluorescent protein DsRed has spectral properties that are ideal for dual-color experiments with green fluorescent protein (GFP)1. But wild-type DsRed has several drawbacks, including slow chromophore maturation and poor solubility2,3. To overcome the slow maturation, we used random and directed mutagenesis to create DsRed variants that mature 10–15 times faster than the wild-type protein. An asparagine-to-glutamine substitution at position 42 greatly accelerates the maturation of DsRed, but also increases the level of green emission. Additional amino acid substitutions suppress this green emission while further accelerating the maturation. To enhance the solubility of DsRed, we reduced the net charge near the N terminus of the protein. The optimized DsRed variants yield bright fluorescence even in rapidly growing organisms such as yeast.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Normalized excitation and emission spectra of representative DsRed variants.
Figure 2: Maturation kinetics of DsRed variants.
Figure 3: Simultaneous visualization of DsRed.


  1. 1

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

    CAS  Article  Google Scholar 

  2. 2

    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 

  3. 3

    Jakobs, S., Subramaniam, V., Schönle, A., Jovin, T.M. & Hell, S.W. EGFP and DsRed expressing cultures of Escherichia coli imaged by confocal, two-photon and fluorescence lifetime microscopy. FEBS Lett. 479, 131–135 (2000).

    CAS  Article  Google Scholar 

  4. 4

    Sullivan, K.F. & Kay, S.A. (eds) Green fluorescent proteins. Vol. 58, Methods in Cell Biology. (Academic Press, San Diego, CA; 1999).

    Google Scholar 

  5. 5

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

    CAS  Article  Google Scholar 

  6. 6

    Cormack, B.P., Valdivia, R.H. & Falkow, S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38 (1996).

    CAS  Article  Google Scholar 

  7. 7

    Heim, R. & Tsien, R.Y. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescent resonance energy transfer. Curr. Biol. 6, 178–182 (1996).

    CAS  Article  Google Scholar 

  8. 8

    Miyawaki, A. et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887 (1997).

    CAS  Article  Google Scholar 

  9. 9

    Wall, M.A., Socolich, M. & Ranganathan, R. The structural basis for red fluorescence in the tetrameric GFP homolog DsRed. Nat. Struct. Biol. 7, 1133–1138 (2000).

    CAS  Article  Google Scholar 

  10. 10

    Yarbrough, D., Wachter, R.M., Kallio, K., Matz, M.V. & Remington, S.J. Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-Å resolution. Proc. Natl. Acad. Sci. USA 98, 462–467 (2001).

    CAS  Article  Google Scholar 

  11. 11

    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 

  12. 12

    Hawley, T.S., Telford, W.G., Ramezani, A. & Hawley, R.G. Four-color flow cytometric detection of retrovirally expressed red, yellow, green, and cyan fluorescent proteins. BioTechniques 30, 1028–1034 (2001).

    CAS  Article  Google Scholar 

  13. 13

    Living Colors™ DsRed2. CLONTECHniques XVI, 2–3 (2001).

  14. 14

    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 

  15. 15

    Wiehler, J., von Hummel, J. & Steipe, B. Mutants of Discosoma red fluorescent protein with a GFP-like chromophore. FEBS Lett. 487, 384–389 (2001).

    CAS  Article  Google Scholar 

  16. 16

    Terskikh, A. et al. “Fluorescent timer”: protein that changes color with time. Science 290, 1585–1588 (2000).

    CAS  Article  Google Scholar 

  17. 17

    Cronin, S. & Hampton, R. A genetics-friendly GFP assay. Trends Cell Biol. 9, 36 (1999).

    Article  Google Scholar 

  18. 18

    Cadwell, R.C. & Joyce, G.F. Mutagenic PCR. In PCR Primer. A laboratory manual. (eds Dieffenbach, C.W. & Dveksler, G.S.) 583–589 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; 1995).

    Google Scholar 

  19. 19

    Verkhusha, V.V. et al. An enhanced mutant of red fluorescent protein DsRed for double labeling and developmental timer of neural fiber bundle formation. J. Biol. Chem. 276, 29621–29624 (2001).

    CAS  Article  Google Scholar 

  20. 20

    Rossanese, O.W. et al. A role for actin, Cdc1p and Myo2p in the inheritance of late Golgi elements in Saccharomyces cerevisiae. J. Cell Biol. 153, 47–61 (2001).

    CAS  Article  Google Scholar 

  21. 21

    Lakowicz, J.R. Principles of fluorescence spectroscopy, Edn. 2. (Kluwer Academic/Plenum Publishers New York, NY; 1999).

    Google Scholar 

  22. 22

    Sikorski, R.S. & Hieter, P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19–27 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Rossanese, O.W. et al. Golgi structure correlates with transitional endoplasmic reticulum organization in Pichia pastoris and Saccharomyces cerevisiae. J. Cell Biol. 145, 69–81 (1999).

    CAS  Article  Google Scholar 

Download references


Thanks to Sergey Lukyanov for sharing unpublished data, to Hiromi Sesaki and Rob Jensen for providing the pCox4-DsRed expression plasmid, to Susan Lindquist for use of the spectrofluorometer, to Dan Strongin for assistance with Supplementary Figure 1A, and to members of the Glick lab for feedback on the manuscript. This work was supported by grants from the National Science Foundation (MCB-9875939) and the American Cancer Society (RPG-00-245-01-CSM).

Author information



Corresponding author

Correspondence to Benjamin S. Glick.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Bevis, B., Glick, B. Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed). Nat Biotechnol 20, 83–87 (2002). https://doi.org/10.1038/nbt0102-83

Download citation

Further reading


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