FluoroCalins represent novel bifunctional protein reagents derived from engineered lipocalins fused to a fluorescent reporter protein, here the enhanced green fluorescent protein (eGFP). We demonstrate the construction, facile bacterial production and broad applicability of FluoroCalins using two Anticalin® molecules directed against the tumor vasculature-associated extra domain B of fibronectin (ED-B) and the vascular endothelial growth factor receptor 3, a marker of tumor and lymphangiogenesis. FluoroCalins were prepared with two different spacers: (i) a short Ser3Ala linker and (ii) a long hydrophilic and conformationally unstructured PASylation® polypeptide comprising 200 Pro, Ala and Ser residues. These FluoroCalins were applied for direct target quantification in enzyme-linked immunosorbent assay as well as target detection by flow cytometry and fluorescence microscopy of live and fixed cells, respectively, demonstrating high specificity and signal-to-noise ratio. Hence, FluoroCalins offer a promising alternative to antibody-based reagents for state of the art fluorescent in vitro detection and biomolecular imaging.

中文翻译：

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FluoroCalins：具有新颖结合功能的工程化脂蛋白，与荧光蛋白融合，可用于生物分子成像和检测。

FluoroCalins代表新颖的双功能蛋白试剂，该蛋白衍生自融合了荧光报告蛋白（此处为增强的绿色荧光蛋白（eGFP））的工程化脂蛋白。我们使用两个针对纤维连接蛋白（ED-B）与肿瘤脉管相关的额外结构域B和血管内皮生长因子受体3（肿瘤和淋巴管生成的标志物）的Anticalin®分子证明了FluoroCalins的构建，简便的细菌生产和广泛的适用性。用两种不同的间隔基制备FluoroCalins：（i）短的Ser3Ala连接子和（ii）包含200个Pro，Ala和Ser残基的长亲水性和构象非结构化的多肽。这些FluoroCalins分别用于酶联免疫吸附测定中的直接靶标定量以及通过活细胞和固定细胞的流式细胞仪和荧光显微镜进行靶标检测，证明了其高特异性和信噪比。因此，FluoroCalins为基于荧光的体外检测和生物分子成像技术提供了一种有希望的替代基于抗体的试剂的方法。