Small monomeric and highly stable near-infrared fluorescent markers derived from the thermophilic phycobiliprotein, ApcF2

摘要

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 (~ 710 nm). They are small monomers (~ 15 kDa) and not only show excellent photostability, but are also thermostable up to 80 °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 . Highlights ? Three near-infrared fluorescent biliproteins (λ Fmax ~ 710 nm), BDFPs, are reported. ? Their labeling is shown in mammalian cells, a nematode and Lactobacillus lactis. ? The BDFPs bind biliverdin covalently and show excellent photostability. ? The BDFPs are thermostable up to 80 °C, tolerate acid down to pH 2. ? Two BDFPs among them are small monomers (~ 15 kDa). ]]>