Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

Summary

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

Abstract

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

Introduction

Protein-protein interactions (PPIs) are critical for the precise regulation of biological processes¹, and further studies on these PPIs can inform about their function². Several approaches have been devised for the study and characterization of PPIs as well as for the de novo identification of novel regulatory protein components. In 1989, Stanley Fields and colleagues reported the yeast two-hybrid (Y2H) assay³. This approach allows for the unbiased and comprehensive identification of interactors (preys) for a defined protein of interest (bait) in Saccharomyces cerevisiae. In addition to its remarkable utility for discovering PPIs, the Y2H assay can be used for characterizing protein pairs in yeast cells, defining minimal interacting domains, and identifying mutations that abolish such interactions. By modifying the Y2H assay, PPIs can also be studied in mammalian cells⁴. Variations of the Y2H assay (e.g., yeast three-hybrid system) can also be applied to study protein-RNA and protein-small organic ligand interactions in cells.

Another commonly used tool to study PPIs in a homologous system is the co-immunoprecipitation (co-IP) assay⁵. By using an antibody to immunoprecipitate a protein of interest, the co-IP assay allows researchers to monitor PPIs in cells for various environmental conditions and experimental situations. The use of epitope-tagged proteins (e.g., FLAG, Myc, STREP, and HA, among others) in affinity purification (AP) methods has facilitated the isolation of proteins from complex protein mixtures for several downstream assays, including western blot, silver stain, and enzymatic analysis. However, none of these previous approaches enable the isolation of large quantities of protein complexes for further characterization including in vitro assays, discovery of regulatory subunits by mass spectrometry, and identification of post-translational modifications. An improved version of the AP method is called Tandem AP (TAP), which is a purification technique for studying PPIs by creating a fusion protein with two epitopes that is purified through two subsequent APs^6,7. In this article, we present a variation of the TAP method for purifying protein complexes in which two subunits are tagged with different epitopes and then purified through two sequential APs (STREP AP followed by FLAG IP). We first provide a minimalistic overview of TAP (Figure 1) and then a detailed description of all the experimental steps (Figure 2), so that researchers can apply them to their protein complex of interest.

To demonstrate the applicability of the TAP method, we chose a well-characterized cyclin-CDK complex (referred to as P-TEFb kinase), which is composed of the regulatory subunit cyclin T1 (CycT1) and a kinase (CDK9), and is involved in the regulation of transcription by RNA polymerase II (Pol II)^8,9,10. P-TEFb phosphorylates the C-terminal domain of Pol II and its associated negative elongation factors, which relieves transcriptional pausing at the promoter and thereby facilitates transcription elongation^11,12,13. With this known interaction in mind, STREP-tagged CycT1 and FLAG-tagged CDK9 were over expressed in HEK293T cells. A reciprocal TAP experiment was performed with STREP-tagged CDK9 and FLAG-tagged CycT1 to further validate that the protein interaction is independent of the epitopes utilized. Cells were collected and lysed 48 h post transfection. The soluble lysate was purified by TAP (STREP AP followed by FLAG IP). Input and purified proteins were analyzed by western blot and silver stain (Figure 3).

Protocol

1. Plating Cells

1. One day prior to transfection, plate 3.5 x 10⁶ HEK293T cells in 10 mL of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/Streptomycin (10,000 U/ml stock) per 100 mm dish. Plate 3 - 5 100 mm dishes per experiment/condition to ensure sufficient protein recovery.
   ​NOTE: The number of plates has to be determined for each experiment/condition, which will rely on the expression levels and solubility of the protein complex of interest. Alternatively, if larger scale purification is needed, cell lines can be adapted to grow in suspensions in spinner flasks², but this method will not work for transient transfections.

2. Transfecting Cells or Inducing Stable Cell Lines 

1. On the following day, check the cells under the microscope. Cells must be 70 - 80% confluent before proceeding.
2. Transfect the cells with a total mixture of 7 µg of plasmid DNA and 21 µl of transfection reagent per 100 mm dish. Transfect cells with a vector expressing GFP (Table 1) as a control for transfection efficiency.
   NOTE: If using an HEK293-T-REx or similar stable cell line that induces the expression of two or more complex subunits in response to doxycycline^14,15, the inducer will have to be added to the cells and incubated for 48 hr. Similarly, a stable cell line that induces the expression of GFP upon the addition of the inducer would also be required for validation purposes. Proceed to step 2.7.
3. Prepare the transfection mixture per 100 mm dish. In a 1.5 ml microcentrifuge tube, mix 500 µl of unsupplemented DMEM with 7 µg of total plasmid DNA (corresponding to two or more plasmids). In a separate microcentrifuge tube, mix 500 µl of unsupplemented DMEM with 21 µl of the transfection reagent. Repeat for each transfection reaction.
4. Pipette the transfection reagent solution into the plasmid DNA solution (do not mix solutions in the reverse order). Mix by pipetting at least 4 times.
5. Incubate this mixture at room temperature for 10 - 15 min, but no longer than 15 min.
6. Tilt the plate to create a media reservoir and carefully pipette the transfection mixture drop-wise onto the interior side of the plate without disturbing the cells. Return the dish to its original flat position and swirl gently to distribute the mixture.
7. Incubate the cells at 37 °C in a humidified cell culture incubator with 5% CO₂ (this condition is hereafter referred to as "tissue culture incubator"). Replace the media 5 hr post-transfection (optional).

3. Checking Transfection or Induction Efficiency

1. At 24 hr post-transfection, visualize the cells under a fluorescent microscope to check for transfection efficiency (or induction of control HEK293T-REx stable cell line expressing GFP). Incubate for another 24 hr.

4. STREP Affinity Purification (STREP AP)

1. Collecting the cells
   1. At 48 hr post-transfection, remove the media and add 8 ml of cold 1x phosphate-buffered saline (PBS). Pipette up and down to detach cells from the plate.
   2. Collect and transfer the cell suspension into a 15 mL tube and spin the cells down at 800 x g for 4 min at 4 °C.
   3. Remove the PBS supernatant. Repeat the spin down (800 x g for 1 min at 4 °C) to eliminate any residual PBS. Store the cell pellet at -80 °C for future use or follow the procedure below to continue protein purification.
2. Lysing the cells
   1. Resuspend the cell pellets using 5× the cell pellet volume (~250 µl per plate) of Passive Lysis Buffer (PLB: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1.5 mM MgCl₂, 1 mM DTT, protease inhibitor cocktail tablet, 5% v/v glycerol, and 1.0% NP-40) and transfer the cell suspension to a 1.5 ml microcentrifuge tube.
   2. Briefly vortex the suspension and nutate for 30 min at 4 °C.
   3. Spin down the cell suspension at 10,000 x g for 10 min at 4 °C.
   4. Transfer the soluble lysates into a new 1.5 ml microcentrifuge tube and discard the cell pellets. Save 10% of the final volume of the soluble lysate as "STREP AP Input." Store at 4 °C.
3. Equilibrating the STREP beads
   NOTE: Use STREP beads for the first AP step! Complexes pulled down with STREP beads recover significantly more protein than those pulled down with FLAG beads.
   1. Take out (n × 40 µl) + n µl of the 50% STREP bead slurry (n = number of samples) and spin down at 1,500 x g for 2 min at 4 °C.
      NOTE: Use wide-mouthed tips to pipette beads.
   2. Remove the supernatant and add 500 µl of PLB to the beads. Nutate the beads mixture for 5 min at 4 °C. Spin down at 1,500 x g for 2 min at 4 °C.
      1. Repeat for a total of 3 washes. Resuspend the beads in PLB to a final volume of n × 40 µl.
   3. Add 40 µl of 50% STREP bead slurry to each cell lysate sample and nutate for 2 hr at 4 °C.
4. STREP AP
   1. Spin down solution at 1,500 x g for 2 min at 4 ˚C.
   2. Remove the supernatant and store as "STREP AP Flow-through (FT)" at 4 °C.
   3. Add 500 µl of STREP AP Wash Buffer I (20 mM Tris-HCl pH 7.5, 250 mM NaCl, 1.5 mM MgCl₂, 1 mM DTT, 5% glycerol and 0.2% NP-40) to the beads. Nutate the beads mixture for 5 min at 4 °C and centrifuge at 1,500 x g for 2 min at 4 °C. Discard the supernatant.
      1. Repeat for a total of 4 washes.
         ​NOTE: Before the fourth wash, transfer the beads solution to a new 1.5 ml microcentrifuge tube to reduce the protein background. This step is essential.
   4. Add 500 µl Wash Buffer II (100 mM Tris-HCl pH 8.0, 150 mM NaCl, and 1 mM EDTA) and equilibrate the beads by inverting the tubes. Spin down at 1,500 x g for 2 min at 4 °C and discard the supernatant.
   5. Elute the protein of interest with 50 µl of STREP Elution Buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, and 2.5 mM Desthiobiotin). Vortex at 800 rpm for 15 min at 4 °C.
   6. Spin down the beads at 1,500 x g for 1 min at 4 °C. Collect the supernatant. This fraction corresponds to the "STREP AP Elution" sample.
   7. Save the beads fraction at 4 °C for a second elution, which could yield up to half of the amount of protein obtained with the first elution. Aliquot 10% of the STREP AP Elution for silver staining and/or western blotting¹⁶.
5. Equilibrating the FLAG beads
   1. Take out (n x 16 µl) + n µl of FLAG beads solution (n = number of samples) and spin down at 1,500 x g for 2 min at 4 °C.
      ​NOTE: Use wide-mouthed tips to pipette beads.
   2. Remove the supernatant and add 500 µL of FLAG wash buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, and 0.1% NP-40) to the beads. Spin down at 1,500 x g for 2 min at 4 °C. Repeat for a total of 3 washes.
   3. Resuspend the beads in FLAG wash buffer to a final volume of n x 16 µl.
   4. Add 16 µl of the FLAG bead slurry to the remaining STREP AP elution and nutate overnight at 4 °C.

5. FLAG IP

1. Spin down the FLAG beads suspension at 1,500 x g for 2 min at 4 °C. Save the supernatant and store as the "FLAG IP Flow-through" (FT) at 4 °C.
2. Add 500 µl of FLAG wash buffer to the solution. Nutate for 5 min at 4 °C and centrifuge at 1,500 x g for 2 min at 4 °C. Repeat for a total of 4 washes and remove the supernatant after each wash.
   NOTE: Before the fourth wash, transfer the protein-beads solution to a new 1.5 ml microcentrifuge tube to reduce background. This step is essential.
3. Remove the supernatant from the final spin and add 30 µL of FLAG wash buffer containing 200 ng/µl of FLAG peptide into the beads. Vortex at 800 rpm for 2 hr at 4 °C.
4. Spin down the suspension at 1,500 x g for 2 min at 4 °C. Harvest the supernatant and store at 4 °C as the "FLAG IP Elution."
   NOTE: The elution samples can be confirmed by silver stain and/or western blot using standard protocols¹⁶, and are ready for further protein assays. When setting up a western blot, run all of the following samples: (1) STREP AP Input, (2) STREP AP FT, (3) STREP AP Elution, (4) FLAG IP FT, and (5) FLAG IP Elution. Volumes loaded will have to be determined for each experiment. All collected samples and beads can be stored at 4 °C for short-term storage and at -80 °C for long-term storage. TAP samples could be analyzed by mass spectrometry to verify the identity of their components, to identify novel post-translational modifications (PTMs), and/or to discover any novel protein interaction with the purified complex^2,17. For this purpose, after running a coomassie gel or silver stain, bands can be excised from the gel using a clean scalpel. Several excellent reviews that discuss mass spectrometry methods were previously published^18,19.

Results

In this article, we demonstrate the applicability of the TAP method to the well-characterized CycT1-CDK9 complex (also known as P-TEFb kinase).

Plasmids encoding Cyclin T1-STREP (CycT1:S) and CDK9-FLAG (CDK9:F), or CDK9-STREP (CDK9:S) and Cyclin T1-FLAG (CycT1:F) (Table 1), were transfected into HEK293T cells. Negative controls included transfections with an empty vector and the CDK9:F or CycT1:F plasmids (

Discussion

The protocol described here for the expression and isolation of protein complexes from eukaryotic cells is not limited to the biochemical characterization of such molecular assemblies, but can also be utilized for the identification of novel interactors and post-translational modifications that could regulate their function. The utilization of affinity tags is not restricted to what is mentioned in this protocol; but our experience suggests that using STREP as the first AP step significantly enhances the yield of the fin...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's Modified Eagle Medium (DMEM)	GE Healthcare Life Sciences/Hyclone	SH3002.2FS
Fetal bovine serum	GE Healthcare Life Sciences/Hyclone	SH30071
Penicillin/Streptomycin	MP Biomedicals	MP091670049
PolyJet	SignaGen Laboratories	SL100688
Protease inhibitor cocktail	Roche	11836153001
STREP-Tactin Superflow	IBA Lifesciences	2-1208-010
STREP-tag elution buffer	IBA Lifesciences	2-1000-025
EZview Red ANTI-FLAG M2 Affinity Gel	Sigma-Aldrich	F2426
Corning 100 mm × 20 mm style dish cell culture treated nonpyrogenic polystyrene 20/sleeve	Corning	430167
Protein Lo-Bind Eppendorf	Eppendorf	022431081
Digital Vortex Mixer	Fisher Scientific	02-215-370
48 hole micro tube foam rack	Fisher Scientific	02-215-386
Labquake shaker rotisserie	Thermo	415110