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...

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	<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. 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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 ...

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