Name: dissecting multi-protein signaling complexes by bimolecular complementation affinity purification (bicap)
Uploaded: 2018-06-15T13:30:00
Description: Watch this Scientific Journal Video about Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP at JoVE.com

D.R.C is a Cancer Institute NSW Fellow and D.N.S was previously a Cancer Institute NSW Fellow. The research findings presented in this manuscript were funded by the Cancer Institute NSW (13/FRL/1-02 and 09/CDF/2-39), NHMRC (Project Grant GNT1052963), Science Foundation Ireland (11/SIRG/B2157), NSW Office of Science and Medical Research, Guest Family Fellowship and Mostyn Family Foundation. J.F.H. and R.S. were recipients of an Australian Postgraduate Award.

1. Plasmid Cloning
NOTE: To generate plasmid vectors with the V1 or V2 tags fused to either the N-terminus or C-terminus of the gene of interest, BiFC destination vectors have been deposited with Addgene [N-terminal tag: pDEST-V1-ORF (#73635), pDEST-V2-ORF (#73636). C-terminal tag: pDEST-ORF-V1 (#73637), pDEST-ORF-V2 (#73638)]. The gene/s of interest will need to be in specific recombination cloning compatible entry vectors (i.e., pDONR223 or pENTR221), without stop codons, to proceed w...

<ol>
	<li>Kolch, W., Pitt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+proteomics+to+dissect+tyrosine+kinase+signalling+pathways+in+cancer.">Functional proteomics to dissect tyrosine kinase signalling pathways in cancer.</a> Nat Rev Cancer. 10 (9), 618-629 (2010).</li><li>Pawson, T., Kofler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinome+signaling+through+regulated+protein-protein+interactions+in+normal+and+cancer+cells.">Kinome signaling through regulated protein-protein interactions in normal and cancer cells.</a> Curr Opin Cell Biol. 21 (2), 147-153 (2009).</li><li>Croucher, D. R., 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=Bimolecular+complementation+affinity+purification+(BiCAP)+reveals+dimer-specific+protein+interactions+for+ERBB2+dimers.">Bimolecular complementation affinity purification (BiCAP) reveals dimer-specific protein interactions for ERBB2 dimers.</a> Sci Signal. 9 (436), ra69 (2016).</li><li>Cassonnet, 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=Benchmarking+a+luciferase+complementation+assay+for+detecting+protein+complexes.">Benchmarking a luciferase complementation assay for detecting protein complexes.</a> Nat Methods. 8 (12), 990-992 (2011).</li><li>Rossi, F., Charlton, C. A., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+protein-protein+interactions+in+intact+eukaryotic+cells+by+beta-galactosidase+complementation.">Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation.</a> Proc Natl Acad Sci U S A. 94 (16), 8405-8410 (1997).</li><li>Magliery, T. J., 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=Detecting+protein-protein+interactions+with+a+green+fluorescent+protein+fragment+reassembly+trap:+scope+and+mechanism.">Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap: scope and mechanism.</a> J Am Chem Soc. 127 (1), 146-157 (2005).</li><li>Hu, C. D., Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+visualization+of+multiple+protein+interactions+in+living+cells+using+multicolor+fluorescence+complementation+analysis.">Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis.</a> Nat Biotechnol. 21 (5), 539-545 (2003).</li><li>Kubala, M. H., Kovtun, O., Alexandrov, K., Collins, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+thermodynamic+analysis+of+the+GFP:GFP-nanobody+complex.">Structural and thermodynamic analysis of the GFP:GFP-nanobody complex.</a> Protein Sci. 19 (12), 2389-2401 (2010).</li><li>Fegan, A., White, B., Carlson, J. C., Wagner, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+controlled+protein+assembly:+techniques+and+applications.">Chemically controlled protein assembly: techniques and applications.</a> Chem Rev. 110 (6), 3315-3336 (2010).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+methods+in+cellular+and+molecular+biology.+plasmid+purification.">Basic methods in cellular and molecular biology. plasmid purification.</a> J Vis Exp. , (2017).</li><li>Eslami, A., Lujan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting:+sample+preparation+to+detection.">Western blotting: sample preparation to detection.</a> J Vis Exp. (44), (2010).</li><li>Shearer, R. F., 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=The+E3+ubiquitin+ligase+UBR5+regulates+centriolar+satellite+stability+and+primary+cilia+formation+via+ubiquitylation+of+CSPP-L.">The E3 ubiquitin ligase UBR5 regulates centriolar satellite stability and primary cilia formation via ubiquitylation of CSPP-L.</a> Mol Biol Cell. , (2018).</li><li>Shannon, 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=Cytoscape:+A+software+environment+for+integrated+models+of+biomolecular+interaction+networks.">Cytoscape: A software environment for integrated models of biomolecular interaction networks.</a> Genome Res. 13 (11), 2498-2504 (2003).</li><li>Schopp, I. 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BiCAP is a powerful method for isolating specific protein dimers while excluding the individual components and their competing binding partners3. BiCAP is based on adaptation of a fluorescence protein complementation assay called BiFC6. Existing methods, including BiFC and proximity ligation assays, have been used extensively to visualize and quantify protein interactions in live cells7, but did not provide an effective means of isolating and charact...

Protein complex assembly is a key process in maintaining the spatiotemporal specificity of many signalling pathways1,2. While the critical nature of this regulatory role is widely recognized, there is a lack of experimental techniques available to scrutinize these complexes. Most interactomics studies focus upon interactions with individual proteins, or the sequential enrichment of individual complex components. Here we present a technique for the isolation of a specific protein dimer while excluding the individual moieties of the component proteins as well as complexes formed with competing binding partners3. We have called this technique Bimolecular Complementation Affinity Purification (BiCAP), as it is a combination of a previously existing protein fragment complementation assay, Bimolecular Fluorescence Complementation (BiFC), with the novel use of a conformation-specific recombinant nanobody towards GFP and its derivatives (see Table of Materials).
A typical protein-fragment complementation assay relies on the expression of &#34;bait&#34; and &#34;prey&#34; proteins fused to split fragments of reporters such as luciferase4, &#946;-galactosidase5, or green fluorescent protein (GFP)6 (Figure 1A). Through the interaction of the bait and prey proteins, the split reporter domains are encouraged to refold into a functional structure, allowing the interaction of the bait and prey proteins to be visualized or quantified. BiCAP was adapted from a version of this technique that made use of fragments of the GFP variant Venus. Fluorescent protein complementation assays are a popular method for visualizing protein-protein interactions in a live cell, but until now have been limited to this one function7. BiCAP represents a significant advance in this regard, as this technique not only allows for visualization, but also the isolation and interrogation of the resulting protein-protein interaction.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57109/57109fig1.jpg" /> 
Figure 1: The structural principal behind the BiCAP technique. (A) A schematic outlining the principal behind bimolecular fluorescence complementation showing the &#39;bait&#39; and &#39;prey&#39; proteins tagged with the N-terminal V1 or C-terminal V2 fragments of the full-length Venus protein. (B) Structural analysis of the interaction interface (cyan) between the GFP nanobody (green) and recombined Venus, showing the position of the V1 (grey) and V2 (orange) fragments (PDB accession 3OGO). This figure is republished fromCroucher et al.3 Reprinted with permission from AAAS. <a href="//cloudflare.jove.com/files/ftp_upload/57109/57109fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The BiCAP technique makes use of two non-fluorescent fragments of Venus (named V1 and V2) which associate with a low degree of affinity unless an interaction occurs between their fusion partners. In this instance, the two split domains refold into the functional &#946;-barrel structure of the fluorophore (Figure 1B)6. The key innovation of BiCAP comes from the introduction of the recombinant GFP nanobody, which recognizes a three-dimensional epitope on the &#946;-barrel of GFP (and variants such as Venus) which is only present on the correctly recombined and folded fluorophore (Figure 1B)8. Crucially, the GFP nanobody does not bind to either of the individual Venus fragments. This facilitates the isolation of protein dimers only after the two proteins have formed a complex of their own volition, leading to more representative results than those acquired from methods that make use of chemically-induced, forced interactions9.
BiCAP is a powerful technique that specifically focuses on multi-protein complexes, which can potentially be combined with a number of downstream applications to improve the granularity of our understanding of the role these complexes play in signal transduction. It also encompasses the important feature of allowing visualization of protein interactions in situ. To date, BiCAP has been demonstrated as an effective method of analyzing the interactome of receptor tyrosine kinase (RTK) dimers3, but the adaptability of this method means that it can be adopted in almost any protein interaction context.

<table>
	<tbody>
		<tr>
			<td>LR Clonase II Plus enzyme</td>
			<td>Thermo Fisher Scientific</td>
			<td>12538120</td>
			<td>Recombinase enzyme required for Gateway cloning (Step 1) into pDEST BiFC destination vectors</td>
		</tr>
		<tr>
			<td>Proteinase K, recombinant, PCR grade</td>
			<td>Thermo Fisher Scientific</td>
			<td>EO0491</td>
			<td></td>
		</tr>
		<tr>
			<td>14 mL round-bottomed polypropylene tube</td>
			<td>Corning</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Roche Diagnostics Australia</td>
			<td>10835242001</td>
			<td>Stock solution prepared at 100 &#956;g/mL in distilled water.</td>
		</tr>
		<tr>
			<td>Miniprep kit</td>
			<td>Promega Corporation</td>
			<td>A1330</td>
			<td></td>
		</tr>
		<tr>
			<td>Maxiprep kit</td>
			<td>Life Technologies Australia</td>
			<td>K2100-07</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11995-073</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Life Technologies Australia</td>
			<td>10099-141</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Life Technologies Australia</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>jetPRIME transfection buffer</td>
			<td>Polyplus</td>
			<td>114-15</td>
			<td></td>
		</tr>
		<tr>
			<td>jetPRIME transfection reagent</td>
			<td>Polyplus</td>
			<td>114-15</td>
			<td></td>
		</tr>
		<tr>
			<td>PhosSTOP (Phosphatase inhibitor)</td>
			<td>Sigma-Aldrich</td>
			<td>4906837001</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete, Mini, EDTA-free Protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Scraper</td>
			<td>Sarstedt</td>
			<td>83.1832</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP-Trap_A</td>
			<td>Chromotek Gmbh</td>
			<td>gta-100</td>
			<td>GFP nanobody coupled to agarose beads</td>
		</tr>
		<tr>
			<td>N-terminal GFP monoclonal antibody&#160;</td>
			<td>Covance</td>
			<td>MMS-118P</td>
			<td>Will detect the V1 tag within the BiFC vectors</td>
		</tr>
		<tr>
			<td>C-terminal GFP monoclonal antibody</td>
			<td>Roche</td>
			<td>11814460001</td>
			<td>Will detect the V2 tag within the BiFC vectors</td>
		</tr>
		<tr>
			<td>Sample buffer</td>
			<td>Invitrogen</td>
			<td>NP0008</td>
			<td>Supplemented with 1 mL &#946;-mercaptoethanol.</td>
		</tr>
		<tr>
			<td>Sequencing grade modified trypsin</td>
			<td>Promega Corporation</td>
			<td>V5117h</td>
			<td></td>
		</tr>
		<tr>
			<td>LoBind microcentrifuge tubes</td>
			<td>Point of Care Diagnostics</td>
			<td>0030 108 116</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Sigma-Aldrich</td>
			<td>I1149-5G</td>
			<td>Prepared at 5 mg/mL in ultrapure water</td>
		</tr>
		<tr>
			<td>Trifluoroacetic Acid - Sequanal Grade</td>
			<td>Thermo Fisher</td>
			<td>10628494</td>
			<td></td>
		</tr>
		<tr>
			<td>3M Empore solid phase extraction C18 disks (octadecyl) - 4.7 cm</td>
			<td>Thermo Fisher</td>
			<td>14-386-2</td>
			<td>To prepare stage tips, cut 1 mm disks using an appropriately sized hole punch. Alternatively, pre-prepared stage tips can also be purchased, see below.</td>
		</tr>
		<tr>
			<td>C18 Stage Tips, 10 &#181;L bed</td>
			<td>Thermo Fisher</td>
			<td>87782</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid OPTIMA for LC/MS grade 50mL</td>
			<td>Thermo Fisher</td>
			<td>FSBA117-50</td>
			<td></td>
		</tr>
		<tr>
			<td>1.9 &#956;m C18 ReproSil particles</td>
			<td>Dr. Maisch GmbH</td>
			<td>r119.aq.</td>
			<td>Stationary phase particles</td>
		</tr>
		<tr>
			<td>Acetonitrile OPTIMA LC/MS grade&#160;</td>
			<td>Thermo Fisher</td>
			<td>FSBA955-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Easy-nLC HPLC&#160;</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LTQ Orbitrap Velos Pro</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Non-ionic detergent (100%)</td>
		</tr>
		<tr>
			<td>DH5&#945; cells</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td>Heat-shock-competent cells</td>
		</tr>
	</tbody>
</table>

dissecting multi-protein signaling complexes by bimolecular complementation affinity purification (bicap)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Following the use of recombination cloning to generate of V1 and V2 tagged genes of interest with the BiFC pDEST plasmids, co-transfection of two plasmids containing an interacting pair of proteins will result in the generation of a Venus fluorescent signal after approximately 8-24 hours. In the absence of a positive signal it is possible that the protein interaction may not be occurring due to the choice of cell line, a low transfection efficiency or that the orientation of the BiFC tags...

Watch this Scientific Journal Video about Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP at JoVE.com

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

This method can help us answer key questions in cell biology and biochemistry, such as how the dynamic assembly of protein and protein complexes control signaling pathways and cell behavior. The main advantage of this technique is that we can specifically isolate two interacting proteins, while excluding uncomplexed individual proteins, and competing binding partners. Though we use this method to provide insight into receptive tyrosine kinase dimerization in breast cancer, it can also be applied to any other physiological setting or disease.We first had an idea for this method when we realized the GFP nanobody recognized the three-dimensional epitope of GFP and other fluorescent proteins. To begin, add 150 nanograms of entry vector, 150 nanograms of destination vector, and eight microliters of TE buffer, to a 0.2 milliliter tube. Next add two microliters of recombinase and ZiMex, and centrifuge the two briefly.Incubate the reaction for one hour at room temperature, then add one microliter of Proteinase K solution, and incubate at 37 degrees celsius for 10 minutes to stop the reaction. Next, thaw heat shock-competent cells on ice. And transfer 50 microliters of the cells to a 14 milliliter round-bottomed polypropylene tube.Add one microliter of reaction product to the cells, and mix gently. Incubate the cells for 20 minutes on ice. Then heat shock the cells in a 42 degree celsius water bath for 45 seconds before returning the cells to the ice.Add one milliliter of LB media to the cells. And incubate the sample with shaking for one hour at 37 degrees celsius. Then, plate the transformation onto a 10 centimeter agar plate with ampicillin, and incubate at 37 degrees celsius overnight.To begin plasmid purification, add ampicillin and 100 milliliters of LB media to a large conical flask. Then use an inoculation loop to remove a single colony from the agar plate. Submerge the inoculation loop in LB media and briefly mix, then cover the top of the flask with aluminum foil, and incubate it overnight, at 37 degrees celsius with shaking.From here purify the plasmid DNA using a standard Maxiprep plasmid DNA purification kit, before proceeding with transfection. First, seed HEK 293T cells in 10 centimeter dishes, containing 10 milliliters of DMEM. Then dilute 2.5 micrograms of each bimolecular fluorescence complementation vector in 500 microliters of transfection buffer.Add 10 microliters of the transfection reagent, and vortex the mixture for 10 seconds. Then briefly centrifuge the samples, and incubate them at room temperature for 10 minutes. Next add the DNA transfection mixture dropwise to the dish.Then incubate the samples for eight to 24 hours. Prepare a cell lysis buffer and supplemented cell lysis buffer as outlined in the text protocol. Then wash the cells with ice cold PBS twice.Aspirate the PBS and add one millimeter of ice-cold supplemented cell lysis buffer. Next, place the dish on ice and incubate for five minutes. Then use a cell scraper to remove the cells, and transfer them to a chilled micro-centrifuge tube.Centrifuge the tube at 18, 000 times gravity for five minutes at four degrees celsius, to remove the cellular debris, then transfer the clear supernatant to a fresh micro-centrifuge tube. Wash an appropriate volume of agarose beads in one milliliter of PBS, then centrifuge the beads at 300 times gravity, and carefully remove the supernatant. Next add 20 microliters of agarose beads to each sample.And incubate at four degrees celsius for two hours, with end-to-end rotation. Centrifuge the beads at 300 times gravity and wash them in cell lysis buffer three times. Then re-suspend the washed beads in 50 microliters of diluted sample buffer.Finally, heat the samples at 95 degrees celsius for two to three minutes. In this study, the BiCAP method is used to observe a positive interaction for several protein types approximately 16 hours after co-transfection of HEK293T cells. Con-focal microscopy is used to observe the dimerization of the RTKERBB2 at the plasma membrane, and the binding of Ubiquitin to the E3 ligase UBR5.After affinity purification with the GFB nanobody, only the full length Venus control protein, and interacting V1 and V2 tagged proteins can be detected in the co-transfection sample. While attempting this procedure, remember to optimize the placement of the bi-molecular fluorescence complementation tag, and either the C-terminal or N-terminal end of your protein of interest. This is necessary to observe a positive protein/protein interaction.After watching this video you should have a good understanding of how to perform the BiCAP technique, and how to apply it to your protein interaction of interest. Following this procedure, many of the downstream characterization techniques could be employed, like ChIPseek to answer additional questions, such as how dimerization of the transcription factors controls DNA binding.

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