Quantitative analysis of gene expression with an improved green fluorescent protein

The fast and easy in vivo detection predestines the green fluorescent prote in (GFP) for its use as a reporter to quantify promoter activities. We have increased the sensitivity of GFP detection 320-fold compared to the wild-t ype by constructing gfp+, which contains mutations improving the folding ef ficiency and the fluorescence yield of GFP+. Twelve expression levels were measured using fusions of the gfp+ and lacZ genes with the tetA promoter in Escherichia coli. The agreement of GFP+ fluorescence with beta-galactosida se activities was excellent, demonstrating that the gfp+ gene can be used t o accurately quantify gene expression in vivo. However, expression of the g fp+ gene from the stronger hsp60 promoter revealed that high cellular conce ntrations of GFP+ caused an inner filter effect reducing the fluorescence b y 50%, thus underestimating promoter activity. This effect is probably due to the higher absorbance of cells containing GFP+. Thus promoters with acti vities differing by about two orders of magnitude can be correctly quantifi ed using the gfp+ gene. Possibilities of using GFP variants beyond this ran ge are discussed.