A novel mutation which enhances the fluorescence of green fluorescent protein at high temperatures

Green fluorescent protein (GFP) from Aequorea victoria is widely used as a marker of gene expression and protein localization in living cells from prokaryotes to eukaryotes. However, the total fluorescent signal from wild-type GFP is very weak when expressed in cells cultured at 37 degrees C compared to 30 degrees C or below. This characteristic makes GFP poorly suited to use as a marker in mammalian cells. Here we describe a new variant of GFP which carries a substitution of Ser147 to Pro (S147P GFP) and which emits a stronger fluorescent signal than the wild-type GFP at high temperature. When S147P is combined with the Ser65 to Thr mutation (S65T GFP), the resulting double mutant emits fluorescence which is several-fold stronger than GFP with a single S65T modification in both bacterial or mammalian cells. This S147P mutation should be useful for constructing new GFP variants which stably emit strong fluorescence at a wide range of culturing temperatures.