A Large Stokes Shift Fluorescent Protein Constructed from the Fusion of Red Fluorescent mCherry and Far‐Red Fluorescent BDFP1.6. (28th March 2019)

• Record Type: Journal Article
• Authors: Zhao, Bao‐Qing; Ding, Wen‐Long; Tan, Zi‐Zhu; Tang, Qi‐Ying; Zhao, Kai‐Hong
• Is Part Of: Chembiochem. Volume 20:Number 9(2019)
• Page Start: 1167
• Page End: 1173
• Publication Date: 2019-03-28
• Subjects: imaging agents -- energy transfer -- fluorescence -- FRET -- proteins; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.201800695
• Languages: English
• ISSNs: 1439-4227

Abstract

Abstract: 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. Abstract : Best of both worlds : BDFP2.0 is a new far‐red fluorescent protein with an extremely high effective brightness and a large Stokes shift, owing to the fusion of BDFP1.6 with mCherry. Thus, BDFP2.0 is not only a good choice as a cell‐component marker, but also an excellent tool in dual‐color imaging for different biological processes.