The first mutant of the Aequorea victoria green fluorescent protein that forms a red chromophore
The first mutant of the Aequorea victoria green fluorescent protein that forms a red chromophore
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DOI：
10.1021/bi702130s
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发表时间：
2008-04-22
期刊：
影响因子：
2.9
通讯作者：
Verkhusha, Vladislav V.
中科院分区：
文献类型：
--
作者：
Mishin, Alexander S.;Subach, Fedor V.;Verkhusha, Vladislav V.
水母及其突变体中的绿色荧光蛋白(GFP)作为荧光标记物被广泛应用于生物医学研究。尽管学术界和工业界为获得其红色荧光突变体做出了巨大的努力，但在克隆以来的15年里，还没有开发出最大发射波长超过529 nm的GFP突变体。在这里，我们使用了一种新的分子进化策略，旨在产生一个绿色荧光蛋白的红色发光突变体。结果，我们成功地获得了第一个GFP突变体，其基本成熟到红光发射状态，最大激发波长和发射波长分别为555和585 nm。该突变体中红色发色团的形成是一种新的、非氧化的机制，包括Ser65侧链的脱水。模型实验表明，绿色和红色荧光的新型双色GFP突变体可以明显地从绿色和红色荧光标记中分离出来，适合作为多色流式细胞术的附加颜色。
Green fluorescent protein (GFP) from a jellyfish, Aequorea victoria, and its mutants are widely used in biomedical studies as fluorescent markers. In spite of the enormous efforts of academia and industry toward generating its red fluorescent mutants, no GFP variants with emission maximum at more than 529 nm have been developed during the 15 years since its cloning. Here, we used a new strategy of molecular evolution aimed at generating a red-emitting mutant of GFP. As a result, we have succeeded in producing the first GFP mutant that substantially matures to the red-emitting state with excitation and emission maxima at 555 and 585 nm, respectively. A novel, nonoxidative mechanism for formation of the red chromophore in this mutant that includes a dehydration of the Ser65 side chain has been proposed. Model experiments showed that the novel dual-color GFP mutant with green and red emission is suitable for multicolor flow cytometry as an additional color since it is clearly separable from both green and red fluorescent tags.