Abstract
Using optimized combinatorial mutagenisis techniques and digital imaging Spectroscopy (DIS), we have insulated mutants of the cloned Aequorea victoria green fluorescent protein (GFP)that show red-shifted excitation spectra similar to that of Renilla reniformis GFP. Selective excitation of wild-type versus Red-Shifted GFP (RSGFP) enables spectral separation of these proteins. Six contiguous codons spanning the tyrosine chromophore region were randomized and sequence analysis of the mutants revealed a tyrosine-glycine consensus. These mutants will enable the simultaneous analysis of two promoters or proteins per cell or organism. In consideration of the multitude of application which are developing for GFP atone, we envisage that spectrally sbtfted fluorescent proteins will be of value to a diversity of research programs, including developmental and cell biology, drug-screening, and diagnostic assays.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Prasher, D.C., Ecbenrode, V.K., Ward, W.W., Prendcrgast, F.G. and Cormier, M.J. 1992. Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111: 229–233.
Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W. and Prasher, D.C. 1994. Green fluorescent protein as a marker for gene expression. Science 263: 802–805.
Ward, W.W. 1979. Energy transfer processes in bioluminescence. Photochem. Photobiol. Rev. 4: 1–57.
Perozzo, M.A., Ward, K.B., Thompson, R.B. and Ward, W.W. 1988. X-ray diffraction and time-resolved fluorescence analyses of Aequorea green-fluorescent protein crystals. J. Biol. Chem. 263: 7713–7716.
Ward, W.W., Cody, C.W. and Hart, R.C. 1980. Spectrophotometric identity of the energy transfer chromophores in Renilla and Aequorea green fluorescent proteins. Photochem. Photobiol. Rev. 31: 611–615.
Shimomura, O. 1979. Structure of the chromophore of Aequorea green fluores cent protein. FEBS Lett. 104: 220–222.
Cody, C.W., Prasher, D.C., Westler, W.M., Prendergast, F.G. and Ward, W.W. 1993. Chemical structure of the hexapeptide chromophore of the Aequorea green fluorescent protein. Biochemistry 32: 1212–1218.
Goldman, E.R. and Youvan, D.C. 1992. An algorithmically optimized combinatorial library screened by digital imaging Spectroscopy. Bio/Technology 10: 1557–1561.
Delagrave, S. and Youvan, D.C. 1993. Searching sequence space to engineer proteins: Exponential ensemble mutagenesis. Bio/Technology 11: 1548–1552.
Hemsley, A., Arnheim, N., Toney, M.D., Cortopassi, G. and Galas, D.J. 1989. A simple method for site-directed mutagenesis using the polymerase chain reaction. Nucleic Acids Research 17: 6546–6551.
Delagrave, S., Goldman, E.R. and Youvan, D.C. 1993. Recursive ensemble mutagenesis. Protein Engineering 6: 327–331.
Inouye, S. and Tsuji, F.I. 1994. Expression of the gene and fluorescence characteristics of the recombinant protein. FEBS Lett. 341: 277–280.
Youvan, D.C., Goldman, E.R., Delagrave, S. and Yang, M.M. 1995. Digital imaging Spectroscopy for massively parallel screening of mutants. Methods in Enzymology 246: 732–748.
Youvan, D.C. 1994. Imaging sequence space. Nature 369: 79–80.
San Pietro, R.M., Prendergast, F.G. and Ward, W.W. 1993. Sequence of the chromogenic hexapeptide of Renilla green-fluorescent protein. Photochem. Photobiol. 57: 63S.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Delagrave, S., Hawtin, R., Silva, C. et al. Red-Shifted Excitation Mutants of the Green Fluorescent Protein. Nat Biotechnol 13, 151–154 (1995). https://doi.org/10.1038/nbt0295-151
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/nbt0295-151
This article is cited by
-
Phosphorylation of tau at Y18, but not tau-fyn binding, is required for tau to modulate NMDA receptor-dependent excitotoxicity in primary neuronal culture
Molecular Neurodegeneration (2017)
-
Recent advances in live cell imaging of hepatoma cells
BMC Cell Biology (2014)
-
Tumor imaging with multicolor fluorescent protein expression
International Journal of Clinical Oncology (2011)
-
Enhanced fluorescent properties of an OmpT site deleted mutant of Green Fluorescent Protein
Microbial Cell Factories (2010)
-
Amphioxus encodes the largest known family of green fluorescent proteins, which have diversified into distinct functional classes
BMC Evolutionary Biology (2009)