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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we present a step-by-step protocol for performing the proximity labeling (PL) experiment in cucumber (Cucumis sativus L.) using AT4G18020 (APRR2)-AirID protein as a model. The method describes the construction of a vector, the transformation of a construct through agroinfiltration, biotin infiltration, protein extraction, and purification of biotin-labeled proteins through affinity purification technique.

Abstract

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA (known as BioID) have proven successful in identifying protein-protein interactions (PPIs). APEX, BioID, and TurboID, a revised version of BioID have some restrictions in addition to being valuable technologies. The recently developed AirID, a novel version of BioID for proximity identification in protein-protein interactions, overcame these restrictions. Previously, AirID has been used in animal models, while the current study demonstrates the use of AirID in plants, and the results confirmed that AirID performs better in plant systems as compared to other PL enzymes such as BioID and TurboID for protein labeling that are proximal to the target proteins. Here is a step-by-step protocol for identifying protein interaction partners using AT4G18020 (APRR2) protein as a model. The methods describe the construction of vector, the transformation of construct through agroinfiltration, biotin transformation, extraction of proteins, and enrichment of biotin-labeled proteins through affinity purification technique. The results conclude that AirID is a novel and ideal enzyme for analyzing PPIs in plants. The method can be applied to study other proteins in plants.

Introduction

Various cellular proteins work under the biologically regulatory system, and protein-protein interactions (PPIs) are a part of this system and the basis of many cellular processes. Besides PPIs, the function of natural proteins is post-translationally promoted via various modifications such as the formation of complex, ubiquitination, and phosphorylation. Therefore, studying PPIs is significant to understanding the possible function of target proteins. PPIs have been carried out using various technologies such as mass spectrometry analysis after immunoprecipitation (IP-MS analysis)1, yeast two-hybrid system (Y2H)2

Protocol

1. Preparation of plant material

  1. Cucumis sativus (Cucumber) is employed for experimental analysis. Put the seeds in water, incubate at 50 °C for 20 min, and then place the seeds on filter paper on a Petri plate for 12-16 h.
  2. Afterward, transfer the seeds to pots containing soil (purchased commercially) and grow in a climatic chamber at 23 °C temperature and 16 h light and 8 h dark photoperiod.
  3. Maintain the plants in the climate chamber for 3-4 weeks until.......

Representative Results

According to previous research, the cucumber gene APRR2 is the candidate gene that controls white immature fruit color8. Here, a protocol was developed using AirID as a proximity labeling enzyme to find the interacting partner protein of APRR2 in cucumber. The construct was transferred to the cucumber leaves, and after 36 h post infiltration, biotin was transferred. After 48 h the samples were taken for western blot analysis to confirm the successful transformation. The proteins .......

Discussion

In the current experiment, AirID was used for proximity labeling, which Kido et al. developed through an algorithm of ancestral enzyme reconstruction using a large genome dataset and five conventional BirA enzymes5. Random mutations were used in traditional evolutionary protein engineering to enhance activity9,10 as random mutations cannot produce dynamic sequence changes. Compared to other PL enzymes, AirID has several advantages. Previou.......

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant No. 32000197 to X.H.), the Special Financial Grant from the China Postdoctoral Science Foundation (Grant No. 2019T120467 to X.H.)

....

Materials

NameCompanyCatalog NumberComments
AcetosyringoneBeijing solaribo science and technology Co.LtdS1519
Acryl/Bis 30% solutionSangon Biotech (Shanghai) Co.Ltd1510KA4528
AgarBioFroxx GmbHD64683
Agarosetsingke (Shanghai) Co.LtdTSJ001
Ammonium bicarbonateSangon Biotech (Shanghai) Co.LtdG313BA0018
BiotinBBI life SciencesG908BA0012
CaCl2BBI life SciencesE209BA0008
Competent cells GV3101Made in the current experiment
Desalting columnThermo scientificWC321753
Deoxycholic acidSangon Biotech (Shanghai) Co.LtdG818BA0029
DH5α competent cellsMade in the current experimentE.coli DH5α
β-D-maltosideBeijing Scolario Science and Tech Co.LtdS818
EDTASangon Biotech (Shanghai) Co.LtdE104BA0029
GlycineSangon Biotech (Shanghai) Co.Ltd161BA0031
HEPESBeijing solaribo science and technology Co.LtdH8090
LiClSangon Biotech (Shanghai) Co.LtdH209BA0003
MESBeijing solaribo science and technology Co.LtdM8019
MiraClothEMD Milipore Corp/MERCK kgAa Darmstadt, Germenay3429963Quick filtration material filter
MgCl2Beijing solaribo science and technology Co.Ltd20200819
NaClSangon Biotech (Shanghai) Co.LtdH324BA0003
NP40Sangon Biotech (Shanghai) Co.LtdN8030
Protein inhibitor cocktailBeijing Scolario Science and Tech Co.LtdS3450
PVDFBIO-RAD5820172
SDSBeijing Scolario Science and Tech Co.LtdS1015
SilwetSangon Biotech (Shanghai) Co.LtdS9430
Streptavidin-C1-conjugated magnetic beadsEnriching Biotechnology7E511E1Magnetic beads
TEMEDServicebioG2056
Triton X-100Sangon Biotech (Shanghai) Co.LtdGB03BA007
Tris-HClSangon Biotech (Shanghai) Co.LtdF828BA0020
TryptoneThermo scientificLP0042

References

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AirIDProtein protein InteractionProximity LabelingCucumis SativusAgrobacterium TumefaciensAgroinfiltrationAPRR2 AirIDPlant BiotechnologyMolecular BiologyProtein Analysis

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