Control of TurboID-dependent biotinylation intensity in proximity ligation screens

Proximity biotinylation screens are a widely used strategy for the unbiased identification of interacting or vicinal proteins. The latest generation biotin ligase TurboID has broadened the range of potential applications, as this ligase promotes an intense and faster biotinylation, even in subcellular compartments like the endoplasmic reticulum. On the other hand, the uncontrollable high basal biotinylation rates deny the system´s inducibility and are often associated with cellular toxicity precluding its use in proteomics. We report here an improved method for TurboID-dependent biotinylation reactions based on the tight control of free biotin levels. Blockage of free biotin with a commercial biotin scavenger reversed the high basal biotinylation and toxicity of TurboID, as shown by pulse-chase experiments. Accordingly, the biotin-blockage protocol restored the biological activity of a bait protein fused to TurboID in the endoplasmic reticulum and rendered the biotinylation reaction inducible by exogenous biotin. Importantly, the biotin-blockage protocol was more effective than biotin removal with immobilised avidin and did not affect the cellular viability of human monocytes over several days. The method presented should be useful to researchers interested in exploiting the full potential of biotinylation screens with TurboID and other high-activity ligases for challenging proteomics questions.

邻近生物素化筛选（proximity biotinylation screens）是实现互作蛋白与邻近蛋白无偏鉴定的通用研究策略。最新一代生物素连接酶TurboID可催化高效且快速的生物素化反应，甚至可在内质网（endoplasmic reticulum）这类亚细胞区室中发挥作用，因此大幅拓展了其潜在应用范围。但该酶存在无法调控的高基础生物素化速率，会导致系统丧失诱导性，且常伴随细胞毒性，使其难以应用于蛋白质组学（proteomics）研究。本研究报道了一种基于严格调控游离生物素水平的改良TurboID依赖型生物素化反应方法。通过商用生物素清除剂阻断游离生物素，可逆转TurboID的高基础生物素化水平与细胞毒性，该结论已通过脉冲追踪实验（pulse-chase experiments）得到验证。据此，该生物素阻断方案可恢复融合有TurboID的诱饵蛋白（bait protein）在内质网中的生物学活性，并使生物素化反应可通过外源添加生物素实现诱导。值得注意的是，该生物素阻断方案相较于使用固定化亲和素去除生物素的方法更为高效，且在数天培养周期内不会影响人单核细胞（human monocytes）的细胞活力。本研究提出的方法，可为希望借助TurboID及其他高活性连接酶充分挖掘生物素化筛选潜力、以解决复杂蛋白质组学问题的研究人员提供有力工具。