Far-red and near-infrared fluorescent proteins have regions of maximum transmission in most tissues and can be widely used as fluorescent biomarkers. We report that fluorescent phycobiliproteins originating from the phycobilisome core subunit ApcF2 can covalently bind biliverdin, named BDFPs. To further improve BDFPs, we conducted a series of studies. Firstly, we mutated K53Q and T144A of BDFPs to increase their effective brightness up to 190% in vivo. Secondly, by homochromatic tandem fusion of high-brightness BDFPs to achieve monomerization, which increases the effective brightness by up to 180% in vivo, and can effectively improve the labeling effect. By combining the above two approaches, the brightness of the tandem BDFPs was much improved compared with that of the previously reported fluorescent proteins in a similar spectral range. The tandem BDFPs were expressed stably while maintaining fluorescence in mammalian cells and Caenorhabditis elegans. They were also photostable and resistant to high temperature, low pH, and chemical denaturation. The tandem BDFPs advantages were proved in applications as biomarkers for imaging in super-resolution microscopy.

中文翻译：

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超高亮度单体远红和近红外荧光胆蛋白

远红和近红外荧光蛋白在大多数组织中具有最大透射区域，可广泛用作荧光生物标记物。我们报道源自藻胆体核心亚基 ApcF2 的荧光藻胆蛋白可以共价结合胆绿素，称为 BDFP。为了进一步改进 BDFP，我们进行了一系列研究。首先，我们对 BDFP 的 K53Q 和 T144A 进行突变，使其体内有效亮度提高至 190%。其次，通过高亮度BDFP的同色串联融合实现单体化，使体内有效亮度提高高达180%，可以有效提高标记效果。通过结合上述两种方法，与之前报道的类似光谱范围内的荧光蛋白的亮度相比，串联BDFP的亮度有了很大的提高。串联 BDFP 在哺乳动物细胞和秀丽隐杆线虫中稳定表达，同时保持荧光。它们还具有光稳定性，耐高温、低 pH 值和化学变性。串联 BDFP 的优势在作为超分辨率显微镜成像生物标志物的应用中得到了证明。