A novel mutant of green fluorescent protein with enhanced sensitivity for microanalysis at 488 nm excitation

Green fluorescent protein (GFP) has been utilized as a powerful reporter of gene expression and protein localization in cells. We discovered a mutant carrying point mutation S208L from a UV-excitable GFP (F99S/M153T/V163A). I t had the enhanced fluorescence intensity. Introduction of the red-shifted mutations (F64L/S65T) to this mutant led to the GFP having the brightest mu tants reported which were expressed in Escherichia coli and excited at 488 nm. The relative fluorescence intensities to that of wild-type GFP and GFPu v were increased about 120- and 10-fold, respectively. It was shown that th e S208L mutation contributes to both a higher intrinsic brightness of GFP a nd a higher expression level in E. coli, (C) 1999 Academic Press.