Red-Shifted Excitation Mutants of the Green Fluorescent Protein
Delagrave S., Hawtin R.E., Silva C.M., Yang M.M., Youvan D.C.
Palo Alto Institute of Molecular Medicine, Mountain View, CA 94043, USA.
Using optimized combinatorial mutagenesis techniques and Digital Imaging Spectroscopy
(DIS), we have isolated 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
applications which are developing for GFP alone, we envisage that spectrally shifted
fluorescent proteins will be of value to a diversity of research programs, including
developmental and cell biology, drug-screening, and diagnostic assays.