36] Generation of a destabilized form of enhanced green fluorescent protein

Abstract

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a useful reporter, widely applied to the study of gene expression and protein localization in vivo. This wide application is because of the autofluorescent activity of the protein, which does not require any substrate or cofactor. Variants with mutations affecting the chromophore structure and the expression of the protein in human cells have been engineered; these mutations have enhanced the expression level and modified the fluorescence properties of GFP in mammalian cells. Enhanced GFP (EGFP) is such a mutant with a 35-fold increase in fluorescence intensity. Its enhanced fluorescence intensity makes EGFP a popular reporter in many applications. By fusion with other proteins, EGFP is widely used to monitor the expression and distribution of the target proteins in live cells. By linking it with a promoter/enhancer element, expression of EGFP can be used to measure the promoter activity as well as to determine its regulation. The introduction of EGFP into transgenic animals permits the study of gene expression and regulation of stage-dependent and spatially distributed genes. The limitation of EGFP as a reporter in certain cases is because of its stability. Crystallographic structures of GFP and its S65T mutant reveal a compacted β-barrel structure. This structure presumably contributes to its unusual resistance to proteolysis and denaturation. The stability of EGFP means a slower turnover in vivo, which limits its applications in certain cases, such as being a reporter in transient transcription studies.