SNAP-tag蛋白标签技术与荧光分析法在蛋白原位分析中的联合应用

为将生物体内微观的蛋白行为可视化并以宏观信号呈现出来对蛋白进行实时、动态分析,借助SNAP-tag蛋白标签技术与有机小分子荧光染料,构建了一系列用于活细胞内实时监测目标蛋白的免洗荧光探针。标签蛋白SNAP-tag能够特异性识别探针中的苄基鸟嘌呤,从而使目标蛋白共价连接上荧光团(萘酰亚胺),携带上荧光信使。此外,由于萘酰亚胺从水环境中被牵引至SNAP-tag蛋白的疏水空腔,其荧光信号呈现出2~13倍的增强。通过SNAP-tag标签蛋白与目标蛋白的融合,该荧光探针实现了对活细胞内线粒体蛋白CoX8A及核内蛋白H2B特异性识别,在免洗条件下完成了对目标蛋白的实时追踪及原位分析。

Combined application of SNAP-tag and fluorescence technique in in-situ protein analysis

The visualization of the microcosmic behavior of proteins in vivo is the key to real-time monitoring of proteins. A series of wash-free SNAP-tag probes were designed and synthesized based on the combination of SNAP-tag and small organic molecule fluorescent dyes. SNAP-tag, which specifically recognized O6-benzylguanine, could be labeled with a fluorophore (e. g., 1,8-naphthalimide) through the formation of covalent bonds. Furthermore, the change from a hydrophilic environment to the hydrophobic cavum of SNAP-tag realized a 2-13-fold enhancement in fluorescence. Through the fusion of SNAP-tag and the target protein, the probes could recognize the mitochondrial proteins (e. g., cytochrome oxidase, Cox8A) and nuclear proteins (e. g., H2B) in living cells. Besides, the fluorescent probes allowed the in-situ real-time monitoring of proteins without washing.