Tandem affinity purification (TAP) technology, established by Rigaut et al. in 1999, relies on molecular cloning techniques to express fusion proteins containing affinity tags in cells. These tags interact with corresponding magnetic beads for the separation and purification of bait proteins.
As the name suggests, tandem purification involves two purification steps. The affinity tag for the fusion protein consists of two parts: Protein A and Tobacco Etch Virus (TEV) protease cleavage site, and a calmodulin binding peptide (CBP). Protein A binds to IgG beads, while CBP binds to calmodulin-coated beads.
After lysing cells containing the fusion protein, the lysate is incubated with IgG beads. The fusion protein and its interacting molecules (proteins or nucleic acids) are retained on the beads. Subsequently, after TEV protease cleavage, the eluate is incubated with calmodulin beads. The interaction between calmodulin and CBP depends on the presence of calcium ions. Finally, elution is performed using a solution containing EGTA to obtain the bait protein and its interacting molecules.
Compared to conventional affinity purification techniques, TAP offers several advantages: (1) Both elution steps are gentle, reducing the loss of molecules interacting with the bait protein. (2) By genetic manipulation, fusion proteins can be expressed under physiological or near-physiological conditions, enabling the study of bait protein interactions in physiological settings. (3) Through two thorough elution steps, the background of the obtained fusion protein and its interacting molecules is low, minimizing false positives.
Due to these advantages, TAP is widely used in the study of protein-protein interactions, particularly in the investigation of protein complexes.
Schematic of the tandem affinity purification procedure (Bailey et al., 2012).
Application of Tandem Affinity Purification Techniques to Yeasts
The initial application of tandem affinity purification (TAP) technology was in yeast systems. Specific sequences were designed on both sides of the fusion gene sequence, and utilizing the high homologous recombination efficiency of yeast, the endogenous bait gene in the yeast chromosome was replaced with a fusion gene carrying affinity tags.
This approach offers several advantages: (1) The fusion gene is expressed under its own promoter, which does not affect the cellular state and allows it to participate in normal physiological activities, thereby ensuring that the obtained interaction information reflects the true physiological processes of the cell. (2) Homologous recombination replaces the endogenous bait gene, eliminating competition from endogenous proteins for interaction partners during the fusion protein's involvement in cellular physiological activities. This enables the acquisition of comprehensive interaction information and high purification efficiency.
It can be considered a nearly perfect strategy. The only flaw, inherent to affinity tag purification techniques, is the potential influence of the tag on protein function. One method to address this issue is to choose a different tag at the N-terminus when the C-terminal tag is not suitable.
In summary, the TAP technology in yeast systems offers a powerful approach for studying protein interactions, providing insights into cellular physiology while overcoming certain limitations inherent in affinity tag purification methods.
Application of Tandem Affinity Purification Techniques to Mammalian Cell Systems
TAP technology has found broad applications across various biological systems, ranging from simple unicellular organisms like yeast to more complex multicellular organisms such as mammals. This technology has been successfully employed in organisms like Escherichia coli, plants, Drosophila, and mammalian cell lines. However, the methodology for implementing TAP differs based on the characteristics of each organism.
In mammalian cells, the expression of fusion proteins for TAP experiments typically involves the construction of fusion genes onto vectors, which are then introduced into the cells using transient or stable transfection methods. Transient transfection often results in high levels of fusion protein expression within a short period. However, such high expression levels can disrupt cellular physiology, leading to potential artifacts and false positives in protein interaction studies.
Mimicking endogenous protein expression levels in mammalian systems poses challenges due to the lower efficiency of homologous recombination compared to yeast. Achieving physiological expression levels of fusion proteins is crucial for accurately studying protein interactions without inducing significant cellular perturbations.
To address the challenges of achieving physiological expression levels, researchers have developed various strategies for stable transfection. These include:
- Establishing stable cell lines based on vectors such as pcDNA3.1 or corresponding vectors, resulting in lower protein expression levels compared to transient transfection.
- Utilizing retroviral vectors such as pZome1N/C or corresponding vectors in retroviral expression systems, which are believed to achieve weak expression levels closer to physiological states.
- Implementing controllable inducible expression systems based on steroid hormones and their receptors, where the expression level of fusion proteins can be adjusted by modulating the amount of steroid hormone analogs (e.g., ponasterone A) added.
To mitigate the effects of competitive interactions from endogenous bait proteins, researchers have combined TAP technology with RNA interference (RNAi). In the iTAP technique, endogenous protein expression in Drosophila is silenced using dsRNA after selecting human proteins as bait. This approach allows for the specific investigation of protein interactions without interference from endogenous proteins.
TAP technology, initially developed and applied in yeast for high-throughput studies, has been adapted for use in mammalian systems. However, the quantification of protein complexes in mammalian systems primarily relies on retroviral expression systems. Despite these challenges, various studies have demonstrated the scalability and effectiveness of TAP technology in unraveling protein interactions and functional networks in mammalian cells.
Experimental Procedure for TAP in Mammalian Systems
Cell Culture:
- Cultivate mammalian cells of interest in appropriate growth media supplemented with serum and antibiotics.
- Maintain cells in a humidified incubator at 37°C with 5% CO2 until reaching approximately 70-80% confluence.
Transfection:
- Prepare fusion constructs containing the protein of interest fused to TAP tags (e.g., Protein A and CBP).
- Transfect mammalian cells with fusion constructs using a suitable transfection reagent according to manufacturer instructions.
- Incubate cells post-transfection for an appropriate period to allow for protein expression.
Protein Extraction:
- Harvest transfected cells by washing with PBS and detaching using trypsin-EDTA.
- Centrifuge cells at 500 × g for 5 minutes and discard the supernatant.
- Wash cell pellets with ice-cold PBS and centrifuge again to obtain a clean cell pellet.
- Lyse cells in an appropriate lysis buffer supplemented with protease inhibitors and RNase inhibitors.
- Incubate cell lysates on ice for 30 minutes with periodic vortexing to ensure complete lysis.
Affinity Purification:
- Prepare Protein A beads by washing with an appropriate buffer and equilibrate with lysis buffer.
- Incubate cell lysates with Protein A beads on a rotator or shaker at 4°C for 2 hours to allow binding of fusion proteins.
- Wash Protein A beads thoroughly with lysis buffer to remove nonspecifically bound proteins.
- Elute bound proteins from Protein A beads using TEV protease cleavage to release the protein of interest and associated partners.
- Collect eluted proteins and proceed to the second affinity purification step.
Second Affinity Purification:
- Prepare calmodulin beads by washing and equilibrating with an appropriate buffer containing calcium ions.
- Incubate eluted proteins from the first step with calmodulin beads at 4°C for 2 hours.
- Wash calmodulin beads to remove nonspecifically bound proteins.
- Elute specifically bound proteins from calmodulin beads using EGTA-containing buffer to disrupt the calcium-dependent interaction.
- Collect eluted proteins for downstream analysis.
Mass Spectrometry Analysis:
- Concentrate eluted protein samples using methods such as acetone precipitation or ultrafiltration.
- Prepare samples for mass spectrometry analysis by trypsin digestion followed by peptide desalting.
- Analyze digested peptides using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify and quantify protein complexes.
- Perform data analysis and interpretation to identify interacting partners and characterize protein complexes.
Reference
- Bailey, Dalan, et al. "Identification of protein interacting partners using tandem affinity purification." JoVE (Journal of Visualized Experiments) 60 (2012): e3643.
