Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer
Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer
复制标题
DOI：
10.1016/s0960-9822(02)00450-5
复制
发表时间：
1996-02-01
期刊：
影响因子：
9.2
通讯作者：
Tsien, RY
中科院分区：
文献类型：
--
作者：
Hein, R;Tsien, RY
背景：不同颜色的绿色荧光蛋白（GFP）变体对于同时比较多种蛋白质命运、发育谱系和基因表达水平非常有用。转移GFP发射颜色最简单的方法是用组氨酸或色氨酸代替发色团中的酪氨酸，但这种蓝移点突变体只能发出微弱的荧光。先前报道的GFP突变体的激发峰和发射峰的最长波长分别为488 nm和511 nm。结果：用组氨酸和色氨酸代替酪氨酸66，主要在145-163残基上进行额外的取代，提高了蓝移GFP突变体的亮度。不同的突变使红移最多的突变体的激发峰和发射峰分别达到504 nm和514 nm。在显微镜下，使用合适的滤镜，至少可以清楚地分辨出三种不同颜色的绿色荧光蛋白突变体。由连接的蓝色和绿色荧光蛋白组成的融合蛋白表现出荧光共振能量转移，这种转移被两个结构域之间的连接物的蛋白水解裂解破坏。结论：我们的结果表明，生产更多更好的绿色荧光蛋白变体是可能的和值得的。这些变异的产生促进了差异基因表达、蛋白质定位或细胞命运的多色成像。不同颜色的突变体之间的融合可能是蛋白酶连续原位测定的有用底物。演示GFP变体之间的能量转移是迈向监测融合蛋白相互关联的通用方法的重要一步。
Background: Variants of the green fluorescent protein (GFP) with different colors would be very useful for simultaneous comparisons of multiple protein fates, developmental lineages and gene expression levels. The simplest way to shift the emission color of GFP is to substitute histidine or tryptophan for the tyrosine in the chromophore, but such blue-shifted point mutants are only dimly fluorescent. The longest wavelengths previously reported for the excitation and emission peaks of GFP mutants are 488 and 511 nm, respectively.Results: Additional substitutions, mainly in residues 145-163, have improved the brightness of the blue-shifted GFP mutants with histidine and tryptophan in place of tyrosine 66. Separate mutations have pushed the excitation and emission peaks of the most red-shifted mutant to 504 and 514 nm, respectively. At least three different colors of GFP mutants can now be cleanly distinguished from each other under the microscope, using appropriate filter sets. A fusion protein consisting of linked blue- and green-fluorescent proteins exhibits fluorescence resonance energy transfer, which is disrupted by proteolytic cleavage of the linker between the two domains.Conclusions: Our results demonstrate that the production of more and better GFP variants is possible and worthwhile. The production of such variants facilitates multicolor imaging of differential gene expression, protein localization or cell fate. Fusions between mutants of different colors may be useful substrates for the continuous in situ assay of proteases. Demonstration of energy transfer between GFP variants is an important step towards a general method for monitoring the mutual association of fusion proteins.