Quantitative analysis of gene expression with an improved green fluorescent protein

Authors: Scholz, Oliver; Thiel, Anja; Hillen, Wolfgang; Niederweis, Michael

Source: FEBS Journal, Volume 267, Number 6, March 2000 , pp. 1565-1570(6)

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Abstract:

The fast and easy in vivo detection predestines the green fluorescent protein (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-type by constructing gfp+, which contains mutations improving the folding efficiency 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 β-galactosidase activities was excellent, demonstrating that the gfp+ gene can be used to accurately quantify gene expression in vivo. However, expression of the gfp+ gene from the stronger hsp60 promoter revealed that high cellular concentrations of GFP+ caused an inner filter effect reducing the fluorescence by 50%, thus underestimating promoter activity. This effect is probably due to the higher absorbance of cells containing GFP+. Thus promoters with activities differing by about two orders of magnitude can be correctly quantified using the gfp+ gene. Possibilities of using GFP variants beyond this range are discussed.

Keywords: gfp; lacZ; reporter gene; fluorescence

Document Type: Research article

DOI: http://dx.doi.org/10.1046/j.1432-1327.2000.01170.x

Publication date: 2000-03-01