TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks
Autor: | Ugrappa Nagalakshmi, Savithramma P. Dinesh-Kumar, Yuan-Yuan Li, Zhiyan Wen, Xinxin Yang, Yongliang Zhang |
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Rok vydání: | 2020 |
Předmět: |
0106 biological sciences
Proteomics Agroinfiltration Protein family purification General Chemical Engineering TurboID 01 natural sciences protein interactions Article General Biochemistry Genetics and Molecular Biology Protein–protein interaction 03 medical and health sciences chemistry.chemical_compound Biotin Affinity chromatography Issue 159 Protein purification Animals Psychology Protein Interaction Maps 030304 developmental biology Immunology and Infection chemistry.chemical_classification 0303 health sciences DNA ligase desalting General Immunology and Microbiology General Neuroscience Plants quantification TIR chemistry Biochemistry Biotinylation Cognitive Sciences Biochemistry and Cell Biology 010606 plant biology & botany proximity labeling |
Zdroj: | Journal of visualized experiments : JoVE, vol 2020, iss 159 J Vis Exp |
ISSN: | 1940-087X |
Popis: | 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 (H(2)O(2)) 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. |
Databáze: | OpenAIRE |
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