一种细胞内快速标记邻近蛋白复合物的新方法

Development of One in Vivo and Rapid Protein-Proximity Labeling Method

目的构建可诱导、细胞适用性广的细胞内快速标记邻近蛋白复合物新方法。方法体外合成TurboID编码基因,并与Tet-on系统一并插入改造后的慢病毒载体,病毒包装后侵染HeLa细胞以融合表达TurboID及目的蛋白YB1或对照蛋白GFP。使用不同浓度的强力霉素处理细胞24h以诱导融合蛋白的表达,加入50μmol/L生物素继续培养15~120min以使TurboID将临近蛋白进行生物素标记,免疫印迹法检测生物素标记的蛋白含量。结果成功构建了可诱导的TurboID融合表达慢病毒载体。获得的病毒侵染HeLa细胞,应用0.125μg/ml以上的强力霉素即可诱导细胞产生TurboID融合蛋白。生物素处理15min以上可得到大量生物素标记的蛋白。结论构建了快速标记邻近蛋白复合物的检测体系——TurboID系统,该系统具有可诱导、生物素标记迅速及细胞适用性广等优点,可广泛应用于大多数动物及人体细胞内蛋白复合物的筛查研究。

Objective To develop an inducible Protein-Proximity Labeling method with rapid and high labeling efficiency in mammalian cells in vivo.Methods The coding sequencing of TurboID gene was synthesized in vitro and inserted into modified lentiviral vector,together with the Tet-on system.After packaging,virus was used to infect HeLa cells for expression of TurboID enzyme,fusing with target protein YB1 or with control protein GFP.Cells were then treated with different concentrations of doxycycline for 24h to induce the expression of these fused proteins.50μmol/L biotin was added and incubated for 15-120min to allow TurboID enzyme to biotinylate proximal proteins in vivo.Then,Western blot was used to detect the biotinylated proteins within treated cells.Results We successfully constructed a lentiviral vector which can be used for the inducible expression of TurboID fused protein.After virus infection,HeLa cells could highly expressed the TurboID fused proteins when treated them with only 0.125μg/ml or more doxycycline.A large amount of biotinylated proteins could also be detected after incubation with biotin for 15min or longer time.Conclusion We successfully developed a rapid Protein-Proximity Labeling method-TurboID system.This system is inducible,biotin labeling highly efficient and cell adaptable.Thus,it can be widely applied for the resolution of protein complexes in most animals and human cells.