Abstract：
BioID is a proximity labeling strategy whose goal is to identify in vivo protein-protein interactions. The central components of this strategy are modified biotin ligase enzymes that promiscuously add biotin groups to proteins in close proximity. The transferred biotin group provides a powerful tag for purification and thus identification of interacting proteins. While a variety of modified biotin ligases were created for BioID, the original enzymes were inefficient, required long incubation times, and high intracellular biotin concentrations for protein labeling. These limitations hinder the application of BioID in contexts such as developing embryos where processes such as cell division and cell fate decisions occur rapidly. Recently, a new biotin ligase called TurboID was developed that addressed many of the deficiencies of previous enzymes. In this paper we compare TurboID to the BioID2 biotin ligase in developing Xenopus embryos. We find that the TurboID enzyme has several advantages over the BioID2 enzyme. TurboID labels proteins efficiently without the addition of additional biotin and occurs at a range of temperatures compatible with the culturing of Xenopus embryos. Biotinylation events occurred rapidly and were limited by TurboID expression and not its activity. Thus, TurboID is an efficient tool for BioID applications in Xenopus embryos and its use should facilitate the identification of interacting proteins in specific networks and complexes during Xenopus development.
摘要：
BioID是一种邻近标记策略，其目标是识别体内蛋白质-蛋白质相互作用。该策略的中心组分是修饰的生物素连接酶,其混杂地将生物素基团添加至紧密接近的蛋白质。转移的生物素基团为纯化提供了强大的标签，从而鉴定了相互作用的蛋白质。虽然为BioID创建了各种修饰的生物素连接酶，原始的酶是低效的，需要较长的孵化时间，和高的细胞内生物素浓度用于蛋白质标记。这些限制阻碍了BioID在诸如发育胚胎的环境中的应用,其中诸如细胞分裂和细胞命运决定的过程迅速发生。最近,开发了一种称为TurboID的新型生物素连接酶，该酶解决了以前酶的许多缺陷。在本文中，我们将TurboID与BioID2生物素连接酶在发育中的非洲爪狼胚胎中进行了比较。我们发现TurboID酶比BioID2酶有几个优点。TurboID在不添加额外的生物素的情况下有效地标记蛋白质,并且在与非洲爪狼胚胎的培养相容的温度范围内发生。生物素化事件迅速发生，并受到TurboID表达而不是其活性的限制。因此，TurboID是BioID应用于非洲爪狼胚胎的有效工具，其使用应有助于在非洲爪狼发育过程中识别特定网络和复合物中的相互作用蛋白。
