Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2003 Jul 15;373(Pt 2):403–408. doi: 10.1042/BJ20021966

A colourless green fluorescent protein homologue from the non-fluorescent hydromedusa Aequorea coerulescens and its fluorescent mutants.

Nadya G Gurskaya 1, Arkady F Fradkov 1, Natalia I Pounkova 1, Dmitry B Staroverov 1, Maria E Bulina 1, Yurii G Yanushevich 1, Yulii A Labas 1, Sergey Lukyanov 1, Konstantin A Lukyanov 1
PMCID: PMC1223499  PMID: 12693991

Abstract

We have cloned an unusual colourless green fluorescent protein (GFP)-like protein from Aequorea coerulescens (acGFPL). The A. coerulescens specimens displayed blue (not green) luminescence, and no fluorescence was detected in these medusae. Escherichia coli expressing wild-type acGFPL showed neither fluorescence nor visible coloration. Random mutagenesis generated green fluorescent mutants of acGFPL, with the strongest emitters found to contain an Glu(222)-->Gly (E222G) substitution, which removed the evolutionarily invariant Glu(222). Re-introduction of Glu(222) into the most fluorescent random mutant, named aceGFP, converted it into a colourless protein. This colourless aceGFP-G222E protein demonstrated a novel type of UV-induced photoconversion, from an immature non-fluorescent form into a green fluorescent form. Fluorescent aceGFP may be a useful biological tool, as it was able to be expressed in a number of mammalian cell lines. Furthermore, expression of a fusion protein of 'humanized' aceGFP and beta-actin produced a fluorescent pattern consistent with actin distribution in mammalian cells.

Full Text

The Full Text of this article is available as a PDF (170.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. 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 U S A. 2000 Oct 24;97(22):11984–11989. doi: 10.1073/pnas.97.22.11984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brejc K., Sixma T. K., Kitts P. A., Kain S. R., Tsien R. Y., Ormö M., Remington S. J. Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein. Proc Natl Acad Sci U S A. 1997 Mar 18;94(6):2306–2311. doi: 10.1073/pnas.94.6.2306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chalfie M. Green fluorescent protein. Photochem Photobiol. 1995 Oct;62(4):651–656. doi: 10.1111/j.1751-1097.1995.tb08712.x. [DOI] [PubMed] [Google Scholar]
  4. Cubitt A. B., Heim R., Adams S. R., Boyd A. E., Gross L. A., Tsien R. Y. Understanding, improving and using green fluorescent proteins. Trends Biochem Sci. 1995 Nov;20(11):448–455. doi: 10.1016/s0968-0004(00)89099-4. [DOI] [PubMed] [Google Scholar]
  5. Ehrig T., O'Kane D. J., Prendergast F. G. Green-fluorescent protein mutants with altered fluorescence excitation spectra. FEBS Lett. 1995 Jun 26;367(2):163–166. doi: 10.1016/0014-5793(95)00557-p. [DOI] [PubMed] [Google Scholar]
  6. Gurskaya N. G., Fradkov A. F., Terskikh A., Matz M. V., Labas Y. A., Martynov V. I., Yanushevich Y. G., Lukyanov K. A., Lukyanov S. A. GFP-like chromoproteins as a source of far-red fluorescent proteins. FEBS Lett. 2001 Oct 19;507(1):16–20. doi: 10.1016/s0014-5793(01)02930-1. [DOI] [PubMed] [Google Scholar]
  7. Heim R., Cubitt A. B., Tsien R. Y. Improved green fluorescence. Nature. 1995 Feb 23;373(6516):663–664. doi: 10.1038/373663b0. [DOI] [PubMed] [Google Scholar]
  8. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  9. Labas Y. A., Gurskaya N. G., Yanushevich Y. G., Fradkov A. F., Lukyanov K. A., Lukyanov S. A., Matz M. V. Diversity and evolution of the green fluorescent protein family. Proc Natl Acad Sci U S A. 2002 Apr 2;99(7):4256–4261. doi: 10.1073/pnas.062552299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lukyanov K. A., Fradkov A. F., Gurskaya N. G., Matz M. V., Labas Y. A., Savitsky A. P., Markelov M. L., Zaraisky A. G., Zhao X., Fang Y. Natural animal coloration can Be determined by a nonfluorescent green fluorescent protein homolog. J Biol Chem. 2000 Aug 25;275(34):25879–25882. doi: 10.1074/jbc.C000338200. [DOI] [PubMed] [Google Scholar]
  11. 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. 1992 Jan;200(1):74–80. doi: 10.1016/0003-2697(92)90279-g. [DOI] [PubMed] [Google Scholar]
  12. Matz M. V., Fradkov A. F., Labas Y. A., Savitsky A. P., Zaraisky A. G., Markelov M. L., Lukyanov S. A. Fluorescent proteins from nonbioluminescent Anthozoa species. Nat Biotechnol. 1999 Oct;17(10):969–973. doi: 10.1038/13657. [DOI] [PubMed] [Google Scholar]
  13. Matz M., Shagin D., Bogdanova E., Britanova O., Lukyanov S., Diatchenko L., Chenchik A. Amplification of cDNA ends based on template-switching effect and step-out PCR. Nucleic Acids Res. 1999 Mar 15;27(6):1558–1560. doi: 10.1093/nar/27.6.1558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Matz Mikhail V., Lukyanov Konstantin A., Lukyanov Sergey A. Family of the green fluorescent protein: journey to the end of the rainbow. Bioessays. 2002 Oct;24(10):953–959. doi: 10.1002/bies.10154. [DOI] [PubMed] [Google Scholar]
  15. Patterson G., Day R. N., Piston D. Fluorescent protein spectra. J Cell Sci. 2001 Mar;114(Pt 5):837–838. doi: 10.1242/jcs.114.5.837. [DOI] [PubMed] [Google Scholar]
  16. Patterson George H., Lippincott-Schwartz Jennifer. A photoactivatable GFP for selective photolabeling of proteins and cells. Science. 2002 Sep 13;297(5588):1873–1877. doi: 10.1126/science.1074952. [DOI] [PubMed] [Google Scholar]
  17. Salih A., Larkum A., Cox G., Kühl M., Hoegh-Guldberg O. Fluorescent pigments in corals are photoprotective. Nature. 2000 Dec 14;408(6814):850–853. doi: 10.1038/35048564. [DOI] [PubMed] [Google Scholar]
  18. Tsien R. Y. The green fluorescent protein. Annu Rev Biochem. 1998;67:509–544. doi: 10.1146/annurev.biochem.67.1.509. [DOI] [PubMed] [Google Scholar]
  19. 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-A resolution. Proc Natl Acad Sci U S A. 2001 Jan 16;98(2):462–467. doi: 10.1073/pnas.98.2.462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. van Thor Jasper J., Gensch Thomas, Hellingwerf Klaas J., Johnson Louise N. Phototransformation of green fluorescent protein with UV and visible light leads to decarboxylation of glutamate 222. Nat Struct Biol. 2002 Jan;9(1):37–41. doi: 10.1038/nsb739. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES