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Ding, Wen-Long; Miao, Dan; Hou, Ya-Nan; Jiang, Su-Ping; Zhao, Bao-Qin; Zhou, Ming; Scheer, Hugo; Zhao, Kai-Hong (2017): Small monomeric and highly stable near-infrared fluorescent markers derived from the thermophilic phycobiliprotein, ApcF2. In: Biochimica et Biophysica Acta-Molecular Cell Research, Vol. 1864, No. 10: pp. 1877-1886
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Abstract

Biliproteins have extended the spectral range of fluorescent proteins into the region of maximal transmission of most tissues and are favorable for multiplexing, but their application presents considerable challenges. Their fluorescence derives from open-chain tetrapyrrole chromophores which often require the introduction of dedicated reductases and lyases. In addition, their fluorescence yield generally decreases with increasing wavelengths and depends strongly on the state of the binding protein. We report fluorescent biliproteins, termed BDFPs, that are derived from the phycobilisome core subunit, ApcF2: this subunit is induced in the thermophilic cyanobacterium, Chroococcidiopsis thermalis, by far-red light and binds phycocyanobilin non-covalently. The BDFPs obtained by molecular evolution of ApcF2 bind the more readily accessible biliverdin covalently while retaining the red-shifted fluorescence in the near-infrared spectral region (similar to 710 nm). They are small monomers (similar to 15 kDa) and not only show excellent photostability, but are also thermostable up to 80 degrees C, tolerate acid down to pH 2 and high concentrations of denaturants. The result indicates far-red adapting cyanobacteria as a useful source for designing extremely red-shifted fluorescent markers. In vivo performance of BDFPs as biomarkers in conventional and super-resolution microscopy, alone or fused to target proteins, is exemplified in several mammalian cells, including, human cell lines, in the nematode, Caenorhabditis elegans and, at low pH, in Lactobacillus lactis.