TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks

生物素化 生物素 蛋白质-蛋白质相互作用 计算生物学 DNA连接酶 生物化学 分子生物学 生物 化学 细胞生物学 DNA
作者
Yongliang Zhang,Yuanyuan Li,Xinxin Yang,Zhiyan Wen,Ugrappa Nagalakshmi,Savithramma P. Dinesh‐Kumar
出处
期刊:Journal of Visualized Experiments [MyJoVE Corporation]
卷期号: (159) 被引量:27
标识
DOI:10.3791/60728
摘要

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been successfully used for identification of protein-protein interactions (PPIs) in mammalian cells. However, requirements of toxic hydrogen peroxide (H2O2) in APEX-based PL, longer incubation time with biotin (16–24 h), and higher incubation temperature (37 °C) in BioID-based PL severely limit their applications in plants. The recently described TurboID-based PL addresses many limitations of BioID and APEX. TurboID allows rapid proximity labeling of proteins in just 10 min under room temperature (RT) conditions. Although the utility of TurboID has been demonstrated in animal models, we recently showed that TurboID-based PL performs better in plants compared to BioID for labeling of proteins that are proximal to a protein of interest. Provided here is a step-by-step protocol for the identification of protein interaction partners using the N-terminal Toll/interleukin-1 receptor (TIR) domain of the nucleotide-binding leucine-rich repeat (NLR) protein family as a model. The method describes vector construction, agroinfiltration of protein expression constructs, biotin treatment, protein extraction and desalting, quantification, and enrichment of the biotinylated proteins by affinity purification. The protocol described here can be easily adapted to study other proteins of interest in Nicotiana and other plant species.
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