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 是一个癌症研究所新南威尔士的研究员和 d.n. S 以前是一个癌症研究所新南威尔士州研究员。本手稿中的研究结果由新南威尔士癌症研究所 (13/FRL/1-02 和 09/民防部队/2-39)、澳洲 (项目赠款 GNT1052963)、爱尔兰科学基金会 (11/SIRG/B2157)、新南威尔士州科学和医学研究办公室、来宾家庭资助。奖学金和 Mostyn 家庭基金会。J.F.H. 和 R.S. 是澳大利亚研究生奖获得者。

1. 质粒克隆
注意: 要生成质粒载体, 将 V1 或 V2 标记融合到感兴趣基因的 N 终点或 C 终点, BiFC 的目标向量已存入 Addgene [N 末端标记: pDEST-V1-ORF (#73635), pDEST-V2-ORF (#73636)。C 终端标签: pDEST-ORF-V1 (#73637), pDEST-ORF-V2 (#73638)]。该基因/s 的兴趣将需要在特定的重组克隆兼容进入媒介 (即pDONR223 或 pENTR221), 没有停止密码子, 以进行克隆。许多相容的克隆已经在各种质粒集合中获得, 包括?...

<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. 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=Split-BioID+a+conditional+proteomics+approach+to+monitor+the+composition+of+spatiotemporally+defined+protein+complexes.">Split-BioID a conditional proteomics approach to monitor the composition of spatiotemporally defined protein complexes.</a> Nat Commun. 8, 15690 (2017).</li></ol>

BiCAP 是一种有效的分离特定蛋白脂肪酸的方法, 而不包括单个成分及其相互竞争的结合伙伴3。BiCAP 是基于一种称为 BiFC6的荧光蛋白互补试验的适应性。现有的方法, 包括 BiFC 和近距离结扎法, 已广泛用于可视化和量化活细胞中的蛋白质相互作用7, 但没有提供一种有效的方法来分离和表征脂肪酸形成的这些相互作用。
基于蛋?...

蛋白质复合组装是维持许多信号通路1、2的时空特异性的关键过程。虽然这一管制作用的关键性质得到广泛承认, 但缺乏用于审查这些复合体的实验技术。大多数 interactomics 研究的重点是与个体蛋白质的相互作用, 或单个复杂成分的顺序浓缩。在这里, 我们提出了一种分离特定蛋白质二聚体的技术, 而不包括单个基团的成分蛋白, 以及形成了与竞争的捆绑合作伙伴3。我们称之为这种技术分子互补亲和纯化 (BiCAP), 因为它是一个以前现有的蛋白质片段互补检测, 分子荧光互补 (BiFC) 的组合, 与新的使用构象特异重组 nanobody 对 GFP 及其衍生物 (见材料表).
一个典型的蛋白质片段互补试验依赖于 "诱饵" 和 "猎物" 蛋白的表达, 融合成分裂的记者, 如荧光素酶4, β-乳糖酶5, 或绿色荧光蛋白 (GFP)6 (图 1A)。通过诱饵和猎物蛋白的相互作用, 鼓励分裂的记者领域再折叠餐巾成一个功能结构, 允许诱饵和猎物蛋白的相互作用被可视化或量化。BiCAP 是根据这种技术的版本改编的, 利用了 GFP 变种金星的片段。荧光蛋白互补检测是一种常见的方法, 以可视化的蛋白质-蛋白质相互作用的活细胞, 但直到现在已被限制在这一功能7。BiCAP 代表了这方面的一个重大进展, 因为这项技术不仅允许可视化, 而且还对由此产生的蛋白质-蛋白质相互作用的隔离和审问。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57109/57109fig1.jpg"> 图 1: BiCAP 技术背后的结构主体.(a) 概述分子荧光互补原理的示意图, 显示 "诱饵" 和 "猎物" 蛋白, 标签为 n-端 V1 或 V2 的全长金星蛋白片段。(B) 对 GFP nanobody (绿色) 与重组金星之间的交互界面 (青色) 进行结构分析, 显示 V1 (灰色) 和 V2 (橙色) 片段 (PDB 加入 3OGO) 的位置。这一数字被重新发布 fromCroucher 等3转载的许可, 由科学促进会。<a href="//cloudflare.jove.com/files/ftp_upload/57109/57109fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
BiCAP 技术利用金星的两个非荧光片段 (命名为 V1 和 V2), 它与低亲和力关联, 除非它们的融合伙伴之间发生交互作用。在此实例中, 两个拆分域再折叠餐巾到荧光的功能β桶结构 (图 1B)6。BiCAP 的关键创新来自于对重组 gfp nanobody 的介绍, 它能识别出 gfp (和金星等变种) 的β桶上的三维表位, 这只存在于正确重组和折叠的荧光 (图 1B)8。关键的是, GFP nanobody 不绑定到单独的金星碎片之一。这促进了蛋白质脂肪酸的分离, 只有在这两种蛋白质形成了自己的意志复杂, 导致更具代表性的结果比从方法, 利用化学诱导, 强迫互动9。
BiCAP 是一种功能强大的技术, 专门专注于多蛋白复合物, 这可能与一些下游应用结合, 以提高我们对这些复合体在信号传导中所起的作用的理解的粒度。.它还包括允许在原位进行蛋白质相互作用可视化的重要特征。迄今为止, BiCAP 已被证明是一种有效的方法来分析受体酪氨酸激酶 (RTK) 脂肪酸3的 interactome, 但这种方法的适应性意味着它可以在几乎任何蛋白质相互作用的情况下采取。

<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)

蛋白质复合体的组装是许多细胞信号通路调控的中心机制。生物医学研究的一个主要重点是破译这些动态蛋白复合物如何将信号从多个来源整合起来, 以指导特定的生物反应, 以及如何在许多疾病的环境中解除管制。尽管这一关键的生化机制的重要性, 缺乏的实验技术, 可以促进具体和敏感的反褶积这些多分子信号复合物。
在这里, 这一缺陷的解决方法是通过蛋白质互补试验与构象特异 nanobody 的结合, 我们称之为分子互补亲和纯化 (BiCAP)。这种新的技术促进了特定的隔离和下游蛋白质组特征的任何一对相互作用的蛋白质, 排除不复杂的个体蛋白和复合物形成与竞争的捆绑合作伙伴。
BiCAP 技术适用于广泛的下游实验测试, 这种技术提供的高度特异性使得对蛋白质复杂装配的力学的更细致的研究比目前可能使用标准亲和纯化技术。

在使用重组克隆产生的 V1 和 V2 标记基因感兴趣的 BiFC pDEST 质粒, 共转染两个质粒含有相互作用对蛋白质将导致产生金星荧光信号后,大约8-24 小时。在没有阳性信号的情况下, 由于细胞系的选择、转染效率低或 BiFC 标记的方向不理想, 蛋白质相互作用可能不会发生。下面将讨论每个方案的疑难解答。
我们以前观察到, 在 HEK-293T 细...

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

分子互补亲和纯化 (BiCAP) 对多蛋白信号络合物的解剖

本手稿描述了分子互补亲和纯化 (BiCAP) 的协议。这种新方法促进了任何两种相互作用的蛋白质的特定隔离和下游蛋白质组特征, 同时排除了不复杂的个体蛋白质以及与竞争的结合伙伴形成的复合体。

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

Research

JoVE Journal

Immunology and Infection

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

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

特定细胞群的净化荧光激活细胞分选（FACS）

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

科研

免疫与感染

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

免疫细胞在受伤的脊髓组织流式细胞仪定量评估：一个细胞的分离纯化方法的新颖使用

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

解剖宿主 - 病毒相互作用模型疱疹病毒裂解性复制

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

热酚水提取纯化及革兰氏阴性菌脂多糖的可视化

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

流感病毒从感染细胞的染色质的核糖核蛋白复合物的亲和纯化

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

病原液泡的分离纯化<em&gt;军团</em&gt;感染吞噬细胞

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

Real-time Live Imaging of T-cell Signaling Complex Formation

T细胞实时成像实时信号复合物的形成

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

细胞因子产生的病毒逃避解剖先天免疫信号

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

细胞与分子极化的快速和强大的分析趋化因子诱导信号

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

测量<em&gt;体外</em&gt; HIV-1 Preintegration配合物的整合活动

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...