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.
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.
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, also cell-free based arrays3. These methods explored various vital findings in the field of research. However, these methods have some drawbacks; for example, Y2H is a time-consuming, expensive strategy that necessitates building the target species' Y2H library.
Additionally, the Y2H technique uses yeast, a heterologous single-cell eukaryotic organism, which could not accurately reflect the cellular state of higher eukaryotic cells. The IP-MS is unsuitable for high hydrophobicity proteins and shows low efficiency in capturing weak PPIs. Various essential proteins in plants such as nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins and receptor-like kinases (RLKs) are expressed at a low level and mostly interact with other proteins transiently; therefore, using these methods is insufficient for understanding the mechanisms underlying the regulation of these proteins3.
A new technique called proximity biotinylation (PB) helps researchers identify PPIs. PB depends on PL enzymes, which attach to the protein of interest (POI), and when partner protein comes near POI, the PL attaches a chemical biotin tag to the partner protein. Further, the tagged protein can be identified and can quickly know which partner protein attaches to the target protein5. Previous studies proved that BioID and TurboID are successful tools for PPIs, especially in plants, but they have certain limitations4. BioID needs a high level of biotin for labeling partner proteins, which takes more than 16 h. Compared to BioID, the TurboID is more beneficial as it labels protein in 10 min and can label the partner protein at room temperature (RT). It is also toxic to cells in certain conditions and tags those proteins that do not show interaction with the protein of interest.
To overcome these issues, AirID, developed by Kido et al., is more efficient than the rest of the labeling enzymes, although the sequence similarity is 82% between BioID and AirID5. To check the efficiency of AirID, we conducted an experiment by using a POI with known associates. This experiment confirmed that AirID could undoubtedly label associated proteins in plant cells. AirID is a valuable enzyme for analyzing PPIs in vitro and in cells. It creates less toxicity and is less erroneous in time taken processes than TurboID to tag non-partners, leading to killing the cell. It demonstrates that AirID is more competitive than other labeling enzymes for proximity biotinylation. It is more accurate, has more potential in time-taking processes, and less toxic in vitro and in living cells. The current protocol describes the identification of interacting proteins of APRR2 using AirID as a PL enzyme; furthermore, the method can be applied to other proteins to investigate PPIs in plant species.
1. Preparation of plant material
2. Making AirID construct
3. Preparation of competent cells
4. Agroinfiltration
5. Collection of samples
NOTE: All materials for sample collection should be sterile to avoid keratin contamination, and all the protocol steps should be performed in a contamination-free environment.
6. Total protein extraction from leaf
7. Equilibrate the desalting column
8. Magnetic beads washing
9. Enrichment of biotinylated proteins
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 were extracted as mentioned above in the protocol for western blot. The representative data in Figure 1 indicated the protein expression and biotinylation in the infiltrated cucumber leaves, which were the expected findings based on the newly modified technique. The results showed a higher expression level of labeled proteins and extra bands as compared to the control. This confirms that AirID successfully tagged the target proteins of the gene of interest (GOI). Further, the results were also confirmed using anti-flag and anti-mouse antibodies, which successfully showed the target band with anti-flag antibody (Figure 2). After confirmation through western blot analysis, we further performed Co-IP using the protocol mentioned above, and the infiltrating leaves' biotinylated proteins were successfully enriched for mass spectrometry analysis, as shown in Figure 3. After enriching biotinylated proteins with streptavidin C1 conjugated magnetic beads, multiple proteins of different sizes were observed, and western blot analysis revealed smeared bands (Figure 3).
Figure 1: Western blot analysis of proteins after transformation to leaves to confirm the function of AirID using streptavidin-HRP. The figure confirms that biotinylated proteins have four independent replicates for the construct transformed samples. Wild-type leaves were used as a control. For each replicate, 10 µL of the samples were loaded for western blot analysis. Streptavidin-HRP (1:6000 dilution) was used to analyze biotinylated proteins in different samples. Please click here to view a larger version of this figure.
Figure 2: Western blot analysis of proteins after transformation to leaves to detect the Taq sequence. The 3x-Flag tag sequence was fused with the gene of interest to confirm the successful transformation. The taq sequence was identified through western blot analysis in all the transformed samples. Wild-type cucumber leaf samples were used as a control. A total 10 µL of the samples were loaded for western blot analysis, and the results were confirmed using anti-flag as a first antibody at dilution of 1:3000 and anti-mouse as a second antibody at dilution 1:10000. Please click here to view a larger version of this figure.
Figure 3: Immunoblot analysis of protein extracts. After Co-immunoprecipitation (Co-IP), Western blot analysis of biotinylated proteins in the agroinfiltrated leaves of Cucumis sativus (Cucumber) with streptavidin-HRP was performed. Please click here to view a larger version of this figure.
Supplementary File 1. Please click here to download this File.
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. Previously this PL method was only applicable to animal systems. In this study, it was used to investigate protein-protein interactions in plants. The protocol outlines how to set up the AirID-based PL in plants step by step, including preparing leaf samples, removing free biotin, quantifying extracted proteins, and enriching biotinylated proteins.
Using the well-established Agrobacterium-mediated transient expression in the Cucumber, this approach is used to find PPIs of the target protein of interest in Cucumber. The transient expression may result in overexpression of the fusion proteins. The AirID fusion must also be tested to ensure that it does not interfere with the function of the target protein of interest, which is another crucial consideration. However, while PL provides several advantages over standard IP-MS techniques for detecting transitory or weak PPIs, it also has some limitations. First and foremost, discovering a candidate interaction protein does not automatically imply a direct or indirect interaction with the bait protein but instead reflects near proximity to the bait protein9. PPIs can be further verified in vivo using independent assays (e.g., co-immunoprecipitation, bimolecular fluorescence complementation (BiFC), or in vitro GST-pull down assay, which can be carried out to verify PPIs further.
The study found that the proximal biotinylation activity of AirID was significantly lower than that of TurboID. In vitro and in cells, TurboID had the highest proximal biotinylation activity. This enzyme may be utilized within 10 min to biotinylate9,11. However, in cells treated for a prolonged incubation of over 6 h and higher biotin concentrations, highest TurboID activity led to extra biotinylation on unexpected proteins, such as tubulin and GFP (such as 50 mM biotin). In contrast to being used as a proximal biotinylation enzyme for PPIs, TurboID was first reported as a biotin-labeling enzyme4,6,5,7. If TurboID were to evaluate PPIs, it would function best under limiting conditions, such as time duration is short, about 1 h in cells. The AirID, biotinylation of tubulin and GFP were not found under the same conditions as those observed for TurboID biotinylation. AirID-fusion proteins were found to be capable of biotinylation of each well-known interactor in both transients, as demonstrated by the streptavidin-pull down assay and the LC-MS/MS analysis5. In low ATP concentrations (1 mM), the formation of biotinoyl-5-AMP was stronger for AirID than for TurboID. It favors lower concentrations of biotin (with 5 mM biotin or without biotin supplement) than TurboID, which prefers higher concentrations. In addition, an examination of biotinylation sites using LC-MS/MS revealed that AirID biotinylation occurred with no unique sequence preference on a nearby Lys residue, indicating that the biotinylation process was non-preferentia5. Although the sequence similarity between BioID and AirID is 82%, AirID showed high biotinylation activity against interacting proteins. Kido et al., 2020 found that AirID can analyze protein-protein interactions in vitro and in cells. Their findings suggest that biotinylation dependent on AirID may be useful for PPI analysis of chemical compounds. The identification of in vivo partners of target proteins is crucial for understanding biological functions12,13, and it has revealed new PPIs, so in vivo proximity biotinylation using BioID has been used in numerous studies. Even when biotin was supplemented, stable expression of AirID did not result in cell-growth inhibition, indicating that the expression of AirID-fusion proteins would be very low-toxic5. AirID is projected to improve PPI-dependent biotinylation accuracy in combination; in summary, it is concluded that AirID is ideal for PPI analysis in plants.
The authors have nothing to disclose.
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.)
Acetosyringone | Beijing solaribo science and technology Co.Ltd | S1519 | |
Acryl/Bis 30% solution | Sangon Biotech (Shanghai) Co.Ltd | 1510KA4528 | |
Agar | BioFroxx GmbH | D64683 | |
Agarose | tsingke (Shanghai) Co.Ltd | TSJ001 | |
Ammonium bicarbonate | Sangon Biotech (Shanghai) Co.Ltd | G313BA0018 | |
Biotin | BBI life Sciences | G908BA0012 | |
CaCl2 | BBI life Sciences | E209BA0008 | |
Competent cells GV3101 | Made in the current experiment | ||
Desalting column | Thermo scientific | WC321753 | |
Deoxycholic acid | Sangon Biotech (Shanghai) Co.Ltd | G818BA0029 | |
DH5α competent cells | Made in the current experiment | E.coli DH5α | |
β-D-maltoside | Beijing Scolario Science and Tech Co.Ltd | S818 | |
EDTA | Sangon Biotech (Shanghai) Co.Ltd | E104BA0029 | |
Glycine | Sangon Biotech (Shanghai) Co.Ltd | 161BA0031 | |
HEPES | Beijing solaribo science and technology Co.Ltd | H8090 | |
LiCl | Sangon Biotech (Shanghai) Co.Ltd | H209BA0003 | |
MES | Beijing solaribo science and technology Co.Ltd | M8019 | |
MiraCloth | EMD Milipore Corp/MERCK kgAa Darmstadt, Germenay | 3429963 | Quick filtration material filter |
MgCl2 | Beijing solaribo science and technology Co.Ltd | 20200819 | |
NaCl | Sangon Biotech (Shanghai) Co.Ltd | H324BA0003 | |
NP40 | Sangon Biotech (Shanghai) Co.Ltd | N8030 | |
Protein inhibitor cocktail | Beijing Scolario Science and Tech Co.Ltd | S3450 | |
PVDF | BIO-RAD | 5820172 | |
SDS | Beijing Scolario Science and Tech Co.Ltd | S1015 | |
Silwet | Sangon Biotech (Shanghai) Co.Ltd | S9430 | |
Streptavidin-C1-conjugated magnetic beads | Enriching Biotechnology | 7E511E1 | Magnetic beads |
TEMED | Servicebio | G2056 | |
Triton X-100 | Sangon Biotech (Shanghai) Co.Ltd | GB03BA007 | |
Tris-HCl | Sangon Biotech (Shanghai) Co.Ltd | F828BA0020 | |
Tryptone | Thermo scientific | LP0042 |