A large Stokes shift fluorescent protein constructed from the fusion of the red fluorescent mCherry and far-red fluorescent BDFP1.6

摘要： While phycobiliproteins are organized as the phycobilisomes, they can harvest orange, red and/or far-red lights for photosynthesis in cyanobacteria and red algae, phycobiliproteins in the phycobilisome cores such as allophycocyanins absorb far-red light for funneling the energy to the reaction centers. Therefore, the allophycocyanin subunits have been engineered as far-red fluorescent proteins such as BDFP1.6. However, most current fluorescent probes have small Stokes shifts, which limits their applications in multicolor bioimaging. mCherry is an excellent fluorescent protein that has maximal emittance in the red spectral range and high fluorescence quantum yield, thus can be used as a donor for energy transfer to the far-red acceptor such as BDFP1.6 via F?rster resonance energy transfer. In this study, mCherry was fused with BDFP1.6, which results in a highly bright far-red fluorescent protein, BDFP2.0, of 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 iRFP670 that is reported as the brightest far-red fluorescent protein. The large Stokes shift of BDFP2.0 facilitates multicolor bioimaging. Therefore, BDFP2.0 can not only well bio-label mammalian including human cells, but also well bio-label the various intracellular components in dual-color imaging.