Fluorescent proteins from nonbioluminescent Anthozoa species

An Erratum to this article was published on 01 December 1999

Abstract

We have cloned six fluorescent proteins homologous to the green fluorescent protein (GFP) from Aequorea victoria. Two of these have spectral characteristics dramatically different from GFP, emitting at yellow and red wavelengths. All the proteins were isolated from nonbioluminescent reef corals, demonstrating that GFP-like proteins are not always functionally linked to bioluminescence. The new proteins share the same β-can fold first observed in GFP, and this provided a basis for the comparative analysis of structural features important for fluorescence. The usefulness of the new proteins for in vivo labeling was demonstrated by expressing them in mammalian cell culture and in mRNA microinjection assays in Xenopus embryos.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: (A) Multiple alignment of fluorescent proteins.
Figure 2: The absorption (solid lines) and emission (dotted lines) spectra for the identified proteins (as determined for the recombinant products of expression in E. coli).
Figure 3: Schematic outline of the fluorophore environment in GFP and its putative variations in new Anthozoa proteins.
Figure 4: Results of synthetic mRNA microinjection assay.

References

  1. 1

    Kendall, J.M. & Badminton, M.N. Aequorea victoria bioluminescence moves into an exciting new era. Trends Biotechnol. 16, 216–224 (1998).

    CAS  Article  Google Scholar 

  2. 2

    Johnson, F.H. et al. Quantum efficiency of Cypridina luminescense, with a note on that of Aequorea. J. Cell. Comp. Physiol. 60, 85–104 (1962).

    CAS  Article  Google Scholar 

  3. 3

    Ward, W.W. in Green fluorescent protein: properties, applications, and protocols. M. Chalfie & S. Kain. 45–75 (Wiley-Liss, New York; 1998).

    Google Scholar 

  4. 4

    Ward, W.W. Energy transfer processes in bioluminescence. Photochem. Photobiol. Rev. 4, 1–57 (1979).

    CAS  Google Scholar 

  5. 5

    Morin, J.G. in Coelenterates biology. Reviews and new perspectives 397–438 (Academic Press, New York; 1974).

    Google Scholar 

  6. 6

    Prasher, D.C. Using GFP to see the light. Trends Genet. 11, 320–323 (1995).

    Article  Google Scholar 

  7. 7

    Labas Y.A . On the mechanism of the calcium activated bioluminescence of Ctenophora. Biophysics of Living Cell 4, 83–110 (1973).

    CAS  Google Scholar 

  8. 8

    Ward, W.W. & Cormier, M.J. An energy transfer protein in coelenterate bioluminescence: characterization of the Renilla green-fluorescent protein. J. Biol. Chem. 254, 781–788 (1979).

    CAS  PubMed  Google Scholar 

  9. 9

    Hastings, J.W. & Morin, J.G. in Green fluorescent protein: properties, applications, and protocols. M. Chalfie & S. Kain. 17–41 (Wiley-Liss, New York; 1998).

    Google Scholar 

  10. 10

    Rees, J.F. et al. The origins of marine bioluminescence—turning oxygen defense-mechanisms into deep-sea communication tools. J. Exp. Biol. 201, 1211–1221 (1998).

    CAS  PubMed  Google Scholar 

  11. 11

    Mazel, C.H. Spectral measurements of fluorescence emission in caribbean cnidarians. Mar. Ecol. Prog. Ser. 120, 185–191 (1995).

    Article  Google Scholar 

  12. 12

    Catala, R. Fluorescence effect from corals irradiated with ultra-violet rays. Nature 183, 949 (1959).

    Article  Google Scholar 

  13. 13

    Kawaguti, S. On the physiology of reef corals. VI. Study of the pigments. Palao trop. Biol. Stn. Stud. 2, 617–674 (1944).

    Google Scholar 

  14. 14

    Schlichter, D., Meier, U. & Fricke, H.W. Improvement of photosynthesis in zooxanthellate corals by autofluorescent chromatophores. Oecologia 99, 124–131 (1994).

    CAS  Article  Google Scholar 

  15. 15

    Delbeek, J.C. & Sprung, J. The reef aquarium: a comprehensive guide to the identification and care of tropical marine invertebrates Vol. 2. (Ricordea Publishing, Coconut Grove, FL; 1994).

    Google Scholar 

  16. 16

    Matz, M. et al. Amplification of cDNA ends based on template-switching effect and step-out PCR. Nucleic Acids Res. 27, 1558–1560 (1999).

    CAS  Article  Google Scholar 

  17. 17

    Ormö, M. et al. Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395 (1996).

    Article  Google Scholar 

  18. 18

    Yang, F., Moss, L.G. & Phillips, G.N. Jr., The molecular structure of green fluorescent protein. Nat. Biotechnol. 14, 1246–1251 (1996).

    CAS  Article  Google Scholar 

  19. 19

    Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10, 1–6 (1997).

    CAS  Article  Google Scholar 

  20. 20

    Cody, C.W., Prasher, D.C., Westler, W.M., Prendergast, F.G. & Ward, W.W. Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein. Biochemistry 32, 1212–1218 (1993).

    CAS  Article  Google Scholar 

  21. 21

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

    CAS  Article  Google Scholar 

  22. 22

    Delgrave, S., Hawtin, R.E., Silva, C.M., Yang, M.M. & Youvan, D.C. Red-shifted excitation mutants of the green fluorescent protein. Bio/Technology 13, 151–154 (1995).

    Google Scholar 

  23. 23

    Chattoraj, M., King, B.A., Bublitz, G.U. & Boxer, S.G. Ultra-fast excited state dynamics in green fluorescent protein: multiple states and proton transfer. Proc. Natl. Acad. Sci. USA 93, 8362–8367 (1996).

    CAS  Article  Google Scholar 

  24. 24

    Brejc, K. et al. Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein. Proc. Natl. Acad. Sci. USA 94, 2306–2311 (1997).

    CAS  Article  Google Scholar 

  25. 25

    Ehrig, T., O'Kane, D.J. & Prendergast, F.G. Green-fluorescent protein mutants with altered fluorescence excitation spectra. FEBS Lett. 367, 163–166 (1995).

    CAS  Article  Google Scholar 

  26. 26

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

    CAS  Article  Google Scholar 

  27. 27

    Miller, A.D. & Rosman, G.J. Improved retroviral vectors for gene transfer and expression Biotechniques 7, 980–990 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

    Zaraisky, A.G. et al. The homeobox-containing gene XANF-1 may control development of the Spemann organizer. Development 121, 3839–3847 (1995).

    CAS  PubMed  Google Scholar 

  29. 29

    Chomczynski, P. & Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159 (1987).

    CAS  Article  Google Scholar 

  30. 30

    Gill, S.C. & Hippel, P.H. Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182, 319–326 (1989).

    CAS  Article  Google Scholar 

  31. 31

    Wetlaufer, D.B. Ultraviolet spectra of proteins and amino acids. Adv. Protein Chem. 17, 303–390 (1962).

    CAS  Article  Google Scholar 

  32. 32

    Mach, H., Middaugh, C.R. & Lewis, R.V. Statistical determination of the average values of the extinction coefficients of tryptophan and tyrosine in native proteins. Anal. Biochem. 200, 74–80 (1992).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We are especially grateful to Andrey Roman'ko, who generously provided the necessary animals from his unique private collection. We thank Dr. Li Ding of Clontech for technical support, and Dr. Andrey Osterman and Dr. Nick Grishin for their critical comments on the manuscript. We also thank Tamara Matz and David Gunn of Clontech for their help in manuscript preparation. This work was supported by the Russian Foundation for Fundamental Research, Clontech Laboratories, and the INTAS-RFBR grant to A.G.Z.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Sergey A. Lukyanov.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Matz, M., Fradkov, A., Labas, Y. et al. Fluorescent proteins from nonbioluminescent Anthozoa species. Nat Biotechnol 17, 969–973 (1999). https://doi.org/10.1038/13657

Download citation

Further reading

Search

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