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.
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