Name: tandem affinity purification of protein complexes from eukaryotic cells
Uploaded: 2017-01-26T14:00:00.000+00:00
Duration: 11 min 30 s
Description: Watch this Scientific Journal Video about Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells at JoVE.com

Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases (NIAID) of the NIH under award number R01AI114362 and Welch Foundation grant I-1782 to Iv&#225;n D'Orso.

1. Plating Cells
<ol>
	<li>One day prior to transfection, plate 3.5 x 106 HEK293T cells in 10 mL of Dulbecco&#39;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. 
	&#8203;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.</li>
</ol>
2. Transfecting Cells or Inducing Stable Cell Lines&#160;
<ol>
	<li>On the following day, check the cells under the microscope. Cells must be 70 - 80% confluent before proceeding.</li>
	<li>Transfect the cells with a total mixture of 7 &#181;g of plasmid DNA and 21 &#181;l 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 doxycycline14,15, 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.</li>
	<li>Prepare the transfection mixture per 100 mm dish. In a 1.5 ml microcentrifuge tube, mix 500 &#181;l of unsupplemented DMEM with 7 &#181;g of total plasmid DNA (corresponding to two or more plasmids). In a separate microcentrifuge tube, mix 500 &#181;l of unsupplemented DMEM with 21 &#181;l of the transfection reagent. Repeat for each transfection reaction.</li>
	<li>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.</li>
	<li>Incubate this mixture at room temperature for 10 - 15 min, but no longer than 15 min.</li>
	<li>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.</li>
	<li>Incubate the cells at 37 &#176;C in a humidified cell culture incubator with 5% CO2 (this condition is hereafter referred to as &#34;tissue culture incubator&#34;). Replace the media 5 hr post-transfection (optional).</li>
</ol>
3. Checking Transfection or Induction Efficiency
<ol>
	<li>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.</li>
</ol>
4. STREP Affinity Purification (STREP AP)
<ol>
	<li>Collecting the cells
	<ol>
		<li>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.</li>
		<li>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 &#176;C.</li>
		<li>Remove the PBS supernatant. Repeat the spin down (800 x g for 1 min at 4 &#176;C) to eliminate any residual PBS. Store the cell pellet at -80 &#176;C for future use or follow the procedure below to continue protein purification.</li>
	</ol>
	</li>
	<li>Lysing the cells
	<ol>
		<li>Resuspend the cell pellets using 5&#215; the cell pellet volume (~250 &#181;l 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.</li>
		<li>Briefly vortex the suspension and nutate for 30 min at 4 &#176;C.</li>
		<li>Spin down the cell suspension at 10,000 x g for 10 min at 4 &#176;C.</li>
		<li>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 &#34;STREP AP Input.&#34; Store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Take out (n &#215; 40 &#181;l) + n &#181;l of the 50% STREP bead slurry (n = number of samples) and spin down at 1,500 x g for 2 min at 4 &#176;C. 
		NOTE: Use wide-mouthed tips to pipette beads.</li>
		<li>Remove the supernatant and add 500 &#181;l of PLB to the beads. Nutate the beads mixture for 5 min at 4 &#176;C. Spin down at 1,500 x g for 2 min at 4 &#176;C.
		<ol>
			<li>Repeat for a total of 3 washes. Resuspend the beads in PLB to a final volume of n &#215; 40 &#181;l.</li>
		</ol>
		</li>
		<li>Add 40 &#181;l of 50% STREP bead slurry to each cell lysate sample and nutate for 2 hr at 4 &#176;C.</li>
	</ol>
	</li>
	<li>STREP AP
	<ol>
		<li>Spin down solution at 1,500 x g for 2 min at 4 &#730;C.</li>
		<li>Remove the supernatant and store as &#34;STREP AP Flow-through (FT)&#34; at 4 &#176;C.</li>
		<li>Add 500 &#181;l 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 &#176;C and centrifuge at 1,500 x g for 2 min at 4 &#176;C. Discard the supernatant.
		<ol>
			<li>Repeat for a total of 4 washes. 
			&#8203;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.</li>
		</ol>
		</li>
		<li>Add 500 &#181;l 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 &#176;C and discard the supernatant.</li>
		<li>Elute the protein of interest with 50 &#181;l 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 &#176;C.</li>
		<li>Spin down the beads at 1,500 x g for 1 min at 4 &#176;C. Collect the supernatant. This fraction corresponds to the &#34;STREP AP Elution&#34; sample.</li>
		<li>Save the beads fraction at 4 &#176;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.</li>
	</ol>
	</li>
	<li>Equilibrating the FLAG beads
	<ol>
		<li>Take out (n x 16 &#181;l) + n &#181;l of FLAG beads solution (n = number of samples) and spin down at 1,500 x g for 2 min at 4 &#176;C. 
		&#8203;NOTE: Use wide-mouthed tips to pipette beads.</li>
		<li>Remove the supernatant and add 500 &#181;L 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 &#176;C. Repeat for a total of 3 washes.</li>
		<li>Resuspend the beads in FLAG wash buffer to a final volume of n x 16 &#181;l.</li>
		<li>Add 16 &#181;l of the FLAG bead slurry to the remaining STREP AP elution and nutate overnight at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
5. FLAG IP
<ol>
	<li>Spin down the FLAG beads suspension at 1,500 x g for 2 min at 4 &#176;C. Save the supernatant and store as the &#34;FLAG IP Flow-through&#34; (FT) at 4 &#176;C.</li>
	<li>Add 500 &#181;l of FLAG wash buffer to the solution. Nutate for 5 min at 4 &#176;C and centrifuge at 1,500 x g for 2 min at 4 &#176;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.</li>
	<li>Remove the supernatant from the final spin and add 30 &#181;L of FLAG wash buffer containing 200 ng/&#181;l of FLAG peptide into the beads. Vortex at 800 rpm for 2 hr at 4 &#176;C.</li>
	<li>Spin down the suspension at 1,500 x g for 2 min at 4 &#176;C. Harvest the supernatant and store at 4 &#176;C as the &#34;FLAG IP Elution.&#34; 
	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 &#176;C for short-term storage and at -80 &#176;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 complex2,17. 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 published18,19.</li>
</ol>

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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KAP1+Recruitment+of+the+7SK+snRNP+Complex+to+Promoters+Enables+Transcription+Elongation+by+RNA+Polymerase+II.">KAP1 Recruitment of the 7SK snRNP Complex to Promoters Enables Transcription Elongation by RNA Polymerase II.</a> Mol Cell. 61, 39-53 (2016).</li><li>Ausubel, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current Protocols in Molecular Biology. , (1994).</li><li>Trudgian, D. 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The authors have nothing to disclose.

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 final protein-protein complex. Both the binding and elution steps to and from the STREP beads are extremely efficient compared with other epitope tags (e.g., FLAG). In addition, in theory, TAP could be applied to other proteins or domains with some minor optimizations, such as buffer conditions, number of plates per experiment (for both transient and stable expressing systems), etc. Furthermore, the TAP method can also be applied to other cell lines if specific protein complexes require a special system for their function or their interactions with unique factors. To facilitate troubleshooting, it is highly recommended that the input and FT samples both be saved (Table 2). When the expected protein bands cannot be visualized in the elution, the FT samples can be used to troubleshoot the experiment and determine whether or not the sample had bound to and/or eluted off of the beads.
A successful TAP is strictly dependent on high levels of protein expression and recovery. Thus, researchers would have to optimize the amount of starting material on a case-by-case basis. If transient transfection is used to express protein components, the transfection efficiency should be at least 70% before proceeding with the second step of the TAP. The reciprocal TAP method (in which the epitopes are swapped between the two baits) usually helps in determining which experimental strategy should be used to achieve the best purity and yield. Again, this will have to be investigated on a case-by-case basis. Importantly, performing reciprocal TAP also confirms that the PPI is independent of the affinity tag used.
Some aspects of the TAP method presented herein have limitations. For example, overexpressing the FLAG- and STREP-tagged proteins could generate artifacts for the ectopically expressed protein itself or their interactors. Different epitope tags may affect protein expression levels, protein conformation, and/or subcellular localization. Besides the cell line and transfection efficiency limitation, the size of the complexes to be purified might be an issue. This TAP method works well for multi-subunit protein complexes. From our experience, four components can be detected by silver stain (data not shown). However, when using two-tagged components, the other interactors may not be co-purified at identical stoichiometric levels. Therefore, for this particular goal, the use of cell lines that induces lower levels of expression of the complex components is advised. Another limitation would be the strength of the PPI. If the interactions are not stable enough, some components could be lost during the purification steps (especially in the final FLAG IP step).
The TAP method was previously described and several different epitope tags were tested, validated, and compared6. However, the TAP method presented in this article is novel and provides an easy, efficient, robust, and alternative strategy to study PPIs, complex assembly, and infer protein network composition and function. Compared with previous TAP methods, this STREP-FLAG TAP strategy allows for the rapid purification of complexes for further characterization without prior knowledge of the complex composition, activity, or function; and without the need of protease cleavage (e.g., TEV) for protein elution (which can significantly decrease protein yield). Because of the increased protein complex recovery, the STREP-FLAG TAP method also allows for the robust identification of proteins that associate with the protein complex of interest in cells through tandem mass spectrometry analysis10,15. Subsequently, other methods, like confocal microscopy, size exclusion chromatography, and co-IP assays, can also be utilized to validate the PPIs. Finally, we propose that an alternative version of the TAP protocol could be adapted to purify and study the assembly of discrete protein-RNA complexes, which will tremendously help researchers in the field of RNA biology.
In summary, we present a method to purify and characterize PPIs in mammalian cell lines. We propose that this method lays the foundation for the future characterization and functional analysis of many novel protein complexes.

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 APs6,7. 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 elongation11,12,13. 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).

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>GE Healthcare Life Sciences/Hyclone</td><td>SH3002.2FS</td><td></td></tr><tr><td>Fetal bovine serum</td><td>GE Healthcare Life Sciences/Hyclone</td><td>SH30071</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>MP Biomedicals</td><td>MP091670049</td><td></td></tr><tr><td>PolyJet</td><td>SignaGen Laboratories</td><td>SL100688</td><td></td></tr><tr><td>Protease inhibitor cocktail&amp;nbsp;</td><td>Roche&amp;nbsp;</td><td>11836153001</td><td></td></tr><tr><td>STREP-Tactin Superflow</td><td>IBA Lifesciences</td><td>2-1208-010</td><td></td></tr><tr><td>STREP-tag elution buffer</td><td>IBA Lifesciences</td><td>2-1000-025</td><td></td></tr><tr><td>EZview Red ANTI-FLAG M2 Affinity Gel</td><td>Sigma-Aldrich</td><td>F2426</td><td></td></tr><tr><td>Corning 100 mm &amp;times; 20 mm style dish cell culture treated nonpyrogenic polystyrene 20/sleeve</td><td>Corning&amp;nbsp;</td><td>430167</td><td></td></tr><tr><td>Protein Lo-Bind Eppendorf</td><td>Eppendorf</td><td>022431081</td><td></td></tr><tr><td>Digital Vortex Mixer</td><td>Fisher Scientific&amp;nbsp;</td><td>02-215-370</td><td></td></tr><tr><td>48 hole micro tube foam rack</td><td>Fisher Scientific</td><td>02-215-386</td><td></td></tr><tr><td>Labquake shaker rotisserie&amp;nbsp;</td><td>Thermo&amp;nbsp;</td><td>415110</td><td></td></tr></tbody></table>

tandem affinity purification of protein complexes from eukaryotic cells

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.

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 (Figures 3A and B, respectively). The proteins were expressed for 48 hr before the cells were collected. Multiple plates of cells were used per experiment (five plates per sample were used for the data presented in Figure 3) to increase protein production and recovery. After the cells were lysed in individual microcentrifuge tubes, the cell lysates of the same sample were combined into one microcentrifuge tube prior to adding the STREP beads. Combining the protein samples with one portion of beads increases the final protein concentration, which can improve protein recovery from proteins that are difficult to express. Following the initial elution, a second elution can be done in order to increase the total protein input for the FLAG IP. It is important to save the input for each purification step before adding the beads for future troubleshooting (Table 2), if needed.
The data in Figure 3A shows the results of the TAP method for CycT1:S and CDK9:F, analyzed by western blot. CDK9:F co-eluted with CycT1:S from the STREP beads, but not in the negative control AP lacking CycT1:S, indicating that CDK9:F and CycT1:S form a protein-protein complex. Expectedly, both proteins were detected in the FLAG elution, which further confirmed the complex formation. Figure 3B shows the results of the reciprocal TAP method, where the epitope tags of the two proteins were swapped (CDK9:S and CycT1:F), and analyzed by western blot.
Figures 3C and 3D show the results of the TAP and reciprocal TAP analyzed by silver staining, respectively. Figure 3C lane 2 showed that CDK9:F co-eluted with CycT1:S off of the STREP beads, but not from the control AP (lane 1), thus indicating that CDK9:F and CycT1:S formed a protein-protein complex. In the subsequent FLAG elution, as shown in lane 4, CycT1:S co-eluted with CDK9:F off of the FLAG beads. Similarly, Figure 3D showed that CycT1:F co-eluted with CDK9:S off of the STREP beads, but not from the control AP, and that the protein-protein complex was efficiently recovered after the second FLAG IP step. Both the western blots and silver stains confirmed that the TAP protocol worked efficiently for the purification, isolation, and characterization of the protein complex of interest.
<img alt="Figure 1" src="/files/ftp_upload/55236/55236fig1.jpg" /> 
Figure 1: Overview of TAP Purification Strategy. A simple scheme describing the steps of TAP. Plasmids encoding the protein of interest with either the STREP or FLAG epitope tag are co-transfected into mammalian cells. After 48 h induction, the cells are collected and lysed. Soluble cell lysate containing the protein of interest along with other proteins are loaded onto STREP beads for STREP AP. Proteins that either contain the STREP tag or are bound to the STREP-tagged protein attached to the beads are then eluted. The FLAG beads were then added into the STREP elution. Proteins that either contain the FLAG tag or are bound to the FLAG-tagged protein attached to the beads are then eluted. Elutions could be analyzed by the indicated methods. <a href="http://cloudflare.jove.com/files/ftp_upload/55236/55236fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55236/55236fig2.jpg" /> 
Figure 2: Schematic Representation of TAP Strategy. A detailed protocol of TAP. See text for details. <a href="http://cloudflare.jove.com/files/ftp_upload/55236/55236fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55236/55236fig3.jpg" />
Figure 3: Example of Step-by-step Analysis of TAP Purification. (A) Western blot analysis of TAP (CycT1:S and CDK9:F). (B) Western blot analysis of reciprocal TAP (CDK9:S and CycT1:F). From 1 ml of whole cell lysate, 5 &#956;l (0.2%) was loaded as &#34;STREP AP Input.&#34; 975 &#956;l of the remaining whole cell lysate was added to STREP beads for STREP AP and 100 &#956;l of &#34;STREP AP Elution&#34; was collected. From this, 5 &#956;l (2%) was loaded. 80 &#956;l of the &#34;STREP AP Elution&#34; was added to FLAG beads for FLAG IP and 30 &#956;l of &#34;FLAG IP Elution&#34; was collected. From the 30 &#956;l of &#34;FLAG IP Elution&#34;, 7 &#956;l (23.3%) was loaded. (C) Silver stain analysis of TAP (CycT1:S and CDK9:F). (D) Silver stain analysis of reciprocal TAP (CDK9:S and CycT1:F). 5 &#956;l (2%) of &#34;STREP AP Elution&#34; and 7 &#956;l (23.3%) of &#34;FLAG IP Elution&#34; were loaded. The asterisks indicate co-eluted impurities. <a href="http://cloudflare.jove.com/files/ftp_upload/55236/55236fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Protein</td>
			<td>Residues</td>
			<td>Vector&#160;</td>
			<td>Tag</td>
			<td>Cloning sites</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td>CycT1</td>
			<td>1-708</td>
			<td>pcDNA/4TO</td>
			<td>STREP</td>
			<td>HindIII - EcoRI</td>
			<td>McNamara et al., 201310</td>
		</tr>
		<tr>
			<td>CycT1</td>
			<td>1-708</td>
			<td>pcDNA/4TO</td>
			<td>FLAG</td>
			<td>HindIII - EcoRI</td>
			<td>This article</td>
		</tr>
		<tr>
			<td>CDK9</td>
			<td>1-372</td>
			<td>pcDNA/4TO</td>
			<td>STREP</td>
			<td>HindIII - XhoI</td>
			<td>This article</td>
		</tr>
		<tr>
			<td>CDK9</td>
			<td>1-372</td>
			<td>pcDNA/4TO</td>
			<td>FLAG</td>
			<td>HindIII - XhoI</td>
			<td>McNamara et al., 201310</td>
		</tr>
		<tr>
			<td>GFP</td>
			<td>1-238</td>
			<td>pcDNA/4TO</td>
			<td>STREP-FLAG</td>
			<td>BamHI - XhoI</td>
			<td>This article</td>
		</tr>
	</tbody>
</table>
Table 1: Plasmids used in this Study. CycT1 and CDK9 were ligated into the pcDNA/4TO-STREP and pcDNA/4TO-FLAG plasmid vectors. These constructs were transformed into DH5&#945; competent cells and plated on LB-Ampicillin plates. Colonies were selected, grown, and screened. Successfully ligated clones were verified by Sanger sequencing and the combination of restriction digestion and agarose gel electrophoresis. For CycT1 we used a shorter version (1 - 708) instead of the full-length (1 - 726) because the last 18 residues contain a PEST sequence (a peptide sequence rich in proline, glutamic acid, serine, and threonine), which acts as a signal peptide for protein degradation20. CDK9 was full-length. The sequence of the tandem STREP tag is as follows: GGGGWSHPQFEKGGGSGGGSGGGSWSHPQFEK. The sequence of the tandem FLAG tag is as follows: GGGGDYKDHDGDYKDHDIDYKDDDDK.
Table 2: Troubleshooting Table. Before beginning with troubleshooting, double check to make sure that each essential step has been followed. Certain steps in this protocol require more optimization than others to achieve the expected result. <a href="http://cloudflare.jove.com/files/ftp_upload/55236/Book2.xlsx">Please click here to download this file.</a>

Watch this Scientific Journal Video about Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells at JoVE.com

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

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.

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

tandem-affinity-purification-protein-complexes-from-eukaryotic

Nanyang Technological University

Research

JoVE Journal

Biochemistry

14.9K Views.  The University of Texas Southwestern Medical Center. 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.

Video: Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Biology

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree of compositional heterogeneity of a complex do not usually impede progress in conducting such studies. In contrast, a complex intended for structural characterization should be purified in a state that is both compositionally and structurally homogeneous as well as at a higher concentration than required for proteomics. Recently, there have been significant advances in the application of electron microscopy for structure determination of large macromolecular complexes. This has heightened interest in approaches to purify native complexes of sufficient quality and quantity for structural determination by electron microscopy. The Tandem Affinity Purification (TAP) method has been optimized to extract and purify an 18-subunit, ~ 0.8 MDa ribonucleoprotein assembly from budding yeast (Saccharomyces cerevisiae) suitable for negative stain and electron cryo microscopy. Herein is detailed the modifications made to the TAP method, the rationale for making these changes, and the approaches taken to assay for a compositionally and structurally homogeneous complex.

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree ...

Tandem Affinity Purification Assay to Study Protein-Protein Interactions

Source: Ma, Z., et al. <a href="https://app.jove.com/v/55236">Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells.</a> J. Vis. Exp. (2017).
This video demonstrates tandem affinity purification &#8212; a technique to purify protein complexes from eukaryotic cells to study protein-protein interaction. The complex contains two proteins labeled with different epitope tags. Upon the purification of the complex using two resins with an affinity for the two different tags in a sequential manner, the presence of both proteins in the elute confirms the interaction between the two.

Tandemowy test oczyszczania powinowactwa do badania interakcji białko-białko

Source: Ma, Z., et al. Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells. J. Vis. Exp. (2017).
This video demonstrates tandem ...

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

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Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Identification of Protein Interacting Partners Using Tandem Affinity Purification

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

The MultiBac Protein Complex Production Platform at the EMBL

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Tandemowy test oczyszczania powinowactwa do badania interakcji białko-białko