IMPROVED FLUORESCENCE AND DUAL-COLOR DETECTION WITH ENHANCED BLUE ANDGREEN VARIANTS OF THE GREEN FLUORESCENT PROTEIN

The green fluorescent protein (GFP) from the jellyfish Aequorea victor ia is a versatile reporter protein for monitoring gene expression and protein localization in a variety of systems, Applications using GFP reporters have expanded greatly due to the availability of mutants wit h altered spectral properties, including several blue emission variant s, all of which contain the single point mutation Tyr-66 to His in the chromophore region of the protein. However, previously described ''BF P'' reporters have limited utility, primarily due to relatively dim fl uorescence and low expression levels attained in higher eukaryotes wit h such variants. To improve upon these qualities, we have combined a b lue emission mutant of GFP containing four point mutations (Phe-64 to Leu, Ser-65 to Thr, Tyr-66 to His, and Tyr-145 to Phe) with a syntheti c gene sequence containing codons preferentially found in highly expre ssed human proteins. These mutations were chosen to optimize expressio n of properly folded fluorescent protein in mammalian cells cultured a t 37 degrees C and to maximize signal intensity. The combination of im proved fluorescence and higher expression levels yield an enhanced blu e fluorescent protein that provides greater sensitivity and is suitabl e for dual color detection with green-emitting fluorophores.