Quantitative analysis of gene expression with an improved green fluorescent protein

被引:161
作者
Scholz, O [1 ]
Thiel, A [1 ]
Hillen, W [1 ]
Niederweis, M [1 ]
机构
[1] Univ Erlangen Nurnberg, Lehrstuhl Mikrobiol, D-91058 Erlangen, Germany
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 2000年 / 267卷 / 06期
关键词
gfp; lacZ; reporter gene; fluorescence;
D O I
10.1046/j.1432-1327.2000.01170.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 beta-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.
引用
收藏
页码:1565 / 1570
页数:6
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