A Large Stokes Shift Fluorescent Protein Constructed from the Fusion of Red Fluorescent mCherry and Far-Red Fluorescent BDFP1.6
A Large Stokes Shift Fluorescent Protein Constructed from the Fusion of Red Fluorescent mCherry and Far-Red Fluorescent BDFP1.6
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红色荧光mCherry和远红色荧光BDFP1.6融合构建的大斯托克斯位移荧光蛋白
DOI：
10.1002/cbic.201800695
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发表时间：
2019
期刊：
影响因子：
3.2
通讯作者：
Zhao Kai-Hong
中科院分区：
文献类型：
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作者：
Zhao Bao-Qing;Ding Wen-Long;Tan Zi-Zhu;Tang Qi-Ying;Zhao Kai-Hong
藻胆蛋白是藻胆体的成分，其可以收获用于蓝藻和红藻中的光合作用的橙子、红色和远红光。藻胆体核心中的藻胆蛋白，如别藻蓝蛋白，吸收远红光以将能量汇集到反应中心。因此，已经将别藻蓝蛋白亚基工程化为远红荧光蛋白，例如BDFP 1.6。然而，目前大多数荧光探针的斯托克斯位移都很小，这限制了它们在生物成像中的应用。mCherry是一种优异的荧光蛋白，其在红色光谱范围内具有最大发射率和高荧光量子产率，因此可以用作供体，通过FRET将能量转移到远红受体，如BDFP 1.6。在这项研究中，mCherry与BDFP 1.6融合，产生了一种高度明亮的远红荧光蛋白BDFP 2.0，具有较大的斯托克斯位移（约79 nm）。 激发能在587 nm处被mCherry最大吸收，并有效地转移到BDFP 1.6;因此在666 nm处最大发射强的远红荧光。 BDFP 2.0在哺乳动物细胞中的有效亮度是iRFP 670的4.2倍，iRFP 670被报道为最亮的远红荧光蛋白。BDFP 2.0的大斯托克斯位移有利于生物成像。因此，BDFP 2.0不仅可以生物标记哺乳动物细胞，包括人类细胞，还可以生物标记双色成像中的各种细胞内成分。
Phycobiliproteins are constituents of phycobilisomes that can harvest orange, red, and far‐red light for photosynthesis in cyanobacteria and red algae. Phycobiliproteins in the phycobilisome cores, such as allophycocyanins, absorb far‐red light to funnel energy to the reaction centers. Therefore, allophycocyanin subunits have been engineered as far‐red fluorescent proteins, such as BDFP1.6. However, most current fluorescent probes have small Stokes shifts, which limit their applications in multicolor bioimaging. mCherry is an excellent fluorescent protein that has maximal emittance in the red spectral range and a high fluorescence quantum yield, and thus, can be used as a donor for energy transfer to a far‐red acceptor, such as BDFP1.6, by FRET. In this study, mCherry was fused with BDFP1.6, which resulted in a highly bright far‐red fluorescent protein, BDFP2.0, with a large Stokes shift (≈79 nm). The excitation energy was absorbed maximally at 587 nm by mCherry and transferred to BDFP1.6 efficiently; thus emitting strong far‐red fluorescence maximally at 666 nm. The effective brightness of BDFP2.0 in mammalian cells was 4.2‐fold higher than that of iRFP670, which has been reported as the brightest far‐red fluorescent protein. The large Stokes shift of BDFP2.0 facilitates multicolor bioimaging. Therefore, BDFP2.0 not only biolabels mammalian cells, including human cells, but also biolabels various intracellular components in dual‐color imaging.