基于TurboID的植物蛋白邻近标记实验方法

Methods for TurboID-based Proximal Labeling in Plants

邻近标记作为近些年发展起来的一项检测活细胞内蛋白互作关系和亚细胞结构蛋白组的新型技术,已成功应用于多种动植物体系的研究。该技术通过给诱饵蛋白融合一个具有特定催化连接活性的酶,在酶的催化作用下将小分子底物(如生物素)共价连接到酶邻近的内源蛋白,通过富集和分析被标记的蛋白可获得与诱饵互作的蛋白组。经定向进化产生的生物素连接酶TurboID具有无蛋白毒性及催化效率高的优势。利用TurboID介导的邻近标记技术分析感兴趣蛋白的邻近蛋白组,可研究细胞内瞬时发生或微弱的蛋白互作网络,进而解析复杂的生物学过程。该文详细描述了在拟南芥(Arabidopsisthaliana)中基于TurboID的邻近标记实验方法及注意事项,旨在为利用这一新技术研究植物蛋白互作关系提供参考。

Proximity labeling(PL), a recently developed technique to detect protein-protein interactions and subcellular structural proteomes in living cells, has been successfully applied in various animal and plant systems. Proximity labeling is conducted by fusing an engineered enzyme with catalytic activity to a protein of interest(bait protein). With the catalysis of the enzyme, small molecular substrates such as biotin are covalently linked to endogenous proximal proteins, which can be further enriched and analyzed to identify the interactome of the bait protein. TurboID, a biotin ligase produced by directed evolution, has the advantages of non-toxicity and high catalytic efficiency. Using TurboID-based proximity labeling to analyze proximal proteins of bait proteins, we can study transient or weak protein interactions, which helps to understand the complex biological processes occurring inside cells. Here, we describe methods and related tips for TurboID-based proximal labeling in Arabidopsis thaliana, and hope to provide a reference for studying plant protein-protein interactions.