Far-red fluorescent tags for protein imaging in living tissues.
Far-red fluorescent tags for protein imaging in living tissues.
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DOI：
10.1042/bj20081949
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发表时间：
2009-03-15
期刊：
影响因子：
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通讯作者：
Chudakov DM
中科院分区：
文献类型：
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作者：
Shcherbo D;Murphy CS;Ermakova GV;Solovieva EA;Chepurnykh TV;Shcheglov AS;Verkhusha VV;Pletnev VZ;Hazelwood KL;Roche PM;Lukyanov S;Zaraisky AG;Davidson MW;Chudakov DM
单体荧光蛋白的一个巨大的调色板已经开发用于研究蛋白质的定位，运动和相互作用。然而，在远红色变体中，低亮度仍然是一个问题，这阻碍了多色标记和全身成像技术。在本文中，我们报道了一种单体远红色荧光蛋白mKate2，它比先前报道的mKate亮近3倍，比mPlum亮10倍。mKate2转基因非洲爪蟾胚胎具有高亮度、远红色发射光谱、优异的耐pH性和光稳定性以及低毒性，使其成为活体组织成像的优越荧光标签。我们还报道了tdKatushka2，这是一种串联远红色标签，在融合中表现良好，提供的近红外荧光比mRaspberry或mCherry亮4倍，比mPlum亮20倍。单体mKate2和伪单体tdKatushka2共同代表了下一代超亮远红色荧光探针，为活细胞和动物蛋白质的荧光成像提供了新的可能性。
A vast colour palette of monomeric fluorescent proteins has been developed to investigate protein localization, motility and interactions. However, low brightness has remained a problem in far-red variants, which hampers multicolour labelling and whole-body imaging techniques. In the present paper, we report mKate2, a monomeric far-red fluorescent protein that is almost 3-fold brighter than the previously reported mKate and is 10-fold brighter than mPlum. The high-brightness, far-red emission spectrum, excellent pH resistance and photostability, coupled with low toxicity demonstrated in transgenic Xenopus laevis embryos, make mKate2 a superior fluorescent tag for imaging in living tissues. We also report tdKatushka2, a tandem far-red tag that performs well in fusions, provides 4-fold brighter near-IR fluorescence compared with mRaspberry or mCherry, and is 20-fold brighter than mPlum. Together, monomeric mKate2 and pseudo-monomeric tdKatushka2 represent the next generation of extra-bright far-red fluorescent probes offering novel possibilities for fluorescent imaging of proteins in living cells and animals.