Properties and Applications of EGFP, Enhanced Color Variants of GFP, and Unstable Derivatives of GFP

Abstract The green fluorescent protein (GFP) has great potential as a tool for biologists because it can be used as an in vivo real time reporter of protein localization and gene expression in a variety of experimental systems. Wild type GFP, however, has several undesirable properties including low brightness, a significant lag in the development of fluorescence, complex photoisomerization, inefficient protein folding at 37°C, and poor expression in several species. To improve upon these qualities, we have combined an ultra-bright variant of GFP, GFPmutl, with a synthetic gene sequence containing codons preferentially found in highly expressed human proteins. The combination of improved fluorescence intensity and higher expression levels yields an enhanced variant (EGFP) (Table 1) that greatly increases the sensitivity of this reporter. The natural green emission of GFP can conveniently be monitored by optics designed to detect fluorescein. There are, however, many potential applications for GFP that require additional emission colors.