Enhanced green fluorescent protein vectors that provide stronger fluorescence intensities and higher expression in mammalian cells

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has become an important reporter molecule for monitoring gene expression and protein localization in vivo and in real time. Unlike other bioluminescent reporters, the chromophore in GFP is intrinsic to the primary structure of the protein allowing GFP fluorescence to be monitored noninvasively in living cells and organisms. Here we describe the isolation of variants of GFP obtained by mutagenesis of the chromophore region of the protein. Fluorescence activated cell sorting (FACS) was used to screen a library of mutant gfp genes in E. coli, and variants of GFP selected which exhibit a 20to 35- fold greater fluorescence intensity when excited at 488 nm. Enhanced fluorescence intensity is due principally to the red-shifted excitation maxima of each variant which range from 488 to 501 nm. Increased fluorescence in bacteria is also attributed to more efficient protein folding relative to wild-type GFP. Expression of GFP has also been increased by tailoring the codon usage of the gene to conform to human codon preferences. We have obtained a significant increase in the sensitivity of GFP in mammalian cells by combining the brightest chromophore mutations with optimized codon usage. These mutants should have wide applicability in studies requiring expression of GFP in combination with detection by standard FITC optics or FACS.