Quantitative analysis of gene expression with an improved green fluorescent protein
Quantitative analysis of gene expression with an improved green fluorescent protein
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DOI：
10.1046/j.1432-1327.2000.01170.x
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发表时间：
2000-03-01
期刊：
影响因子：
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通讯作者：
Niederweis, M
中科院分区：
文献类型：
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作者：
Scholz, O;Thiel, A;Niederweis, M
绿色荧光蛋白（GFP）的快速和简单的体内检测决定了其用作定量启动子活性的报告基因。我们通过构建含有提高GFP+的折叠效率和荧光产率的突变的gfp+，将GFP检测的灵敏度提高了320倍。使用大肠杆菌中的tetA启动子的gfp+和lacZ基因的融合物测量12个表达水平。GFP+荧光与β-半乳糖苷酶活性的一致性非常好，表明gfp+基因可用于准确定量体内基因表达。然而，从更强的hsp 60启动子的gfp+基因的表达揭示，高细胞浓度的GFP+引起内过滤器效应，使荧光降低50%，从而低估了启动子活性。这种效应可能是由于含有GFP+的细胞的吸光度较高。因此，可以使用gfp+基因正确地定量具有相差约两个数量级的活性的启动子。讨论了使用超出此范围的GFP变体的可能性。
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.