Development of One in Vivo and Rapid Protein-Proximity Labeling Method
Liu Jian;Zhou Shan;Dai Dapeng;The MOH Key Laboratory of Geriatrics, Beijing Hospital,National Center of Gerontology;
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 24 h to induce the expression of these fused proteins. 50μmol/L biotin was added and incubated for 15-120 min 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 15 min 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.