Name: functional reconstitution and channel activity measurements of purified wildtype and mutant cftr protein
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Description: Watch this Scientific Journal Video about Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein at JoVE.com

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1.净化CFTR的注意：请参见表材料和设备的设备和材料在这个协议中使用的列表。存在重量的人，CFTR的过度表达和突变体在S F 9杆状病毒表达系统17,28的详细协议。过度CFTR并准备了S F 9细胞根据此协议的颗粒。 <ol><li>粗膜制备<ol><li>获取新的或从500毫升培养细胞过表达wildtype-，F508del-或G551D-CFTR蛋白的C末端His 10 -tag的解冻冷冻S F 9细胞沉淀，?...

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已经出现了纯化流程为全长，功能性CFTR隔离数量有限，从各种细胞过表达系统。此处所描述的方法是有利的，因为它允许将Wt-CFTR或F508del-和G551D-CFTR的中等量的高富集的快速纯化即高功能的测定中，包括ATP酶和通道功能的直接测量，包括在平面双层单信道测量系统和人口CFTR通道功能表现的措施是高度相关的小分子19-21的研究。纯化方法被集成到一个快速有效的重建协议，从其他的方法但是...

跨越上皮细胞在这样的组织如肺，肠，胰腺和汗腺的顶膜的氯离子转运主要由囊性纤维化跨膜传导调节因子（CFTR），一个ATP-和磷酸化调节的部件的基础知识（A​​TP-介导的结合膜蛋白的录像带）C亚科（1回顾）。像ABCC亚科的其他成​​员，CFTR是一个大的，多生成树整合膜蛋白，其在形成在其核苷酸结合结构域（NBDS），在那里它具有适度的ATP酶活性，在一个单一的接口两个核苷酸结合位点结合的ATP站点。然而，与其他ABCC亚家族成员，CFTR已演变为一种独特的监管氯通道而不是作为一个活跃的溶质转运。 CFTR突变导致囊性纤维化，这种疾病影响多个器官包括肺，胃肠道，胰腺和生殖道，导致morbidity和死亡率在青壮年。肺部疾病通常占在囊性纤维化早期死亡率和在大多数情况下是由CFTR功能上的传导气道表面上皮的损失。 CFTR氯离子通道活性的缺乏导致在两个氯的减少-横跨表面上皮和水的运动来修改流体层上的纤毛呼吸上皮的顶端表面。这导致粘稠的气道表面液体，可以削弱纤毛呼吸道上皮细胞的能力，以有效地清除病原体从气道。其结果是，大部分的CF患者患有肺部感染和肺损伤引起炎症的反复发作。 正如预期的那样，正常的CFTR蛋白的作用机制研究主要集中在其通道门控活动的详细的电生理研究。单信道研究表明直接说CFTR用作PKA依赖性Cl-它拥有一个ATP调节的2号门通道。详细的电生理学研究提供了大量的关于单个CFTR通道1,3的信息，但是可能有关心是否已被研究的任何特定单个信道的特性是反射性CFTR通道的全部人口，因此单信道的结果应始终与方法来研究宏观的人口考虑沿。纯化CFTR的人口通道活性的直接检测有可能提供洞察与致病突变相关的分子缺陷，并带动化工发现其调制器修复突变CFTR蛋白。迄今为止，有超过在CFTR 1900不同的突变被认为造成囊性纤维化4。主要的突变，F508del-CFTR，发现至少一个等位基因的患者，在北美和欧洲的约90％会导致蛋白质错误折叠一第二保留于内质网5。 F508del-CFTR也有其他后果，包括有缺陷的通道活性6-9。从细胞表面所得到的不存在的CFTR的与严重的疾病有关。 G551D-CFTR，一个不太常见的突变，被认为是正确折叠但是不正常如在细胞表面6一氯离子通道。小分子校正和增效剂的发展具有校正的折叠和/或贩卖的突变体，如F508del-CFTR到细胞表面的，和增效或增加突变的通道活性如G551D在细胞表面上存在时，目标分别。而校正的VX-809和VX-661（尚未批准使用的患者，所述增效剂Kalydeco（ivacaftor; VX-770）正在使用，在150毫克每12小时在CF患者&gt; 6年与至少一个G551D -CFTR突变，以及最近的患者G178R，S549N，S549R，G551S，G1244E，S1251N，S1255P之一和G1349D。 Kalydeco是既安全又导致改善的疾病的CF 10的临床措施，这种小分子的作用却是知之甚少在FDA批准用于在患者使用时的机制。 少数CFTR纯化方法先前已被描述2,11-18，其中许多需要相当长的时间才能完成。在最近的出版物19，一个独特的快速纯化和重组方法被用于过度表达的CFTR中的S F 9细胞表达系统描述的，并且在所定义的脂质系统本纯化的蛋白被用来建立一个CFTR卤化物通道活性测定为CFTR的人口分子。该测定概括的磷酸，核苷酸和抑制剂对CFTR功能的已知作用。该系统被用来询问增强剂的效果的VX-770 / Kalydeco上重量（野生型），F508del-和G551D-CFTR和它表明用于第一时间，该药物直接交互的CFTR蛋白，以使可能在一个ATP无关的方式其信道活动，从总体角度来证明这些方法的实用性和适用性CFTR与核苷酸和小分子的突变体的相互作用的研究回答关于蛋白质临床相关的问题。该方法也被用来研究其它增强剂分子和它们的衍生物20中 ，以及对蛋白质21的活性的小分子校正的效果。 流出测定法已被用于在许多研究中预先调查的CFTR突变体的活性和CFTR-调节化合物在其活性的影响，包括使用的电极，放射性示踪剂和荧光团22,23的全细胞测定法，膜囊，用离子选择性电极24和纯化重组CFTR与放射性示踪剂25。然而THE使用的离子选择性电极，研究纯化重组CFTR首次最近19日报道。当前方法的改编已用于重建和绿脓杆菌 ，一个共同的CF病原体2的膜蛋白的功能特性。加之碘化流出测量纯化ALGE外膜蛋白的重组被用来通过此传送机构26，以支持一个模型阴离子藻分泌。重构和碘化流出测量施加到纯化WZX蛋白，其允许被提出了一个模型，通过该蛋白27表明一个H +依赖性反向转运机制为跨细菌内膜的脂质连接寡糖的易位。在这两种情况下，碘化用作替代物的阴离子基底，尽管在较低的吞吐量比人们所预料要天然底物。该方法可以适用于适应其它蛋白质与Cationic运输或传导途径阴离子基底。 这里一个快速纯化过程被描述为CFTR蛋白和其重组到所定义的脂质脂蛋白体。迅速重建过程可以很容易地定制用于与CFTR通过其他方法纯化的使用，其前提是在纯化中使用的洗涤剂的种类是适合于除去由这里所使用的方法，也可以用于重建过程之前合适的去污剂交换。碘化物流出方法纯化和重组CFTR蛋白的通道活性的测定进行详细说明，并可以从该方法得到的一些典型结果。

<table border="0" cellpadding="0" cellspacing="0" width="1269">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:403px;">fos-choline 14 detergent</td>
			<td style="width:515px;">Anatrace Affymetrix (www.anatrace.affymetrix.com)</td>
			<td style="width:145px;">F312</td>
			<td style="width:207px;">Affymetrix: Anatrace Products; CAS#&#160;:77733-28-9</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cOmplete EDTA-free protease inhibitor cocktail tablets</td>
			<td>Roche (www.roche-applied-science.com)</td>
			<td>04 693 132 001</td>
			<td>EDTA-free Protease inhibitor cocktail tablets (1 in 50 ml or mini:1 in 10 mL) (Roche Diagnostics GmbH; Ref: 11873 580 001</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpack&#160;</td>
			<td>Roche (www.roche-applied-science.com)</td>
			<td>05 892 791 001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ni-NTA Agarose</td>
			<td>Qaigen GmbH</td>
			<td align="right">1018240</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fisherbrand Screening columns</td>
			<td>Fisher Healthcare</td>
			<td>11-387-50</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Amicon Ultra Centrifugal filters, Ultracel-100K</td>
			<td>Millipore (www.millipore.com)</td>
			<td>UFC910008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cAMP-Dependent Protein Kinase A (PKA), Catalytic Subunit</td>
			<td colspan="2">New England Biolabs (www.neb.com/products/p6000-camp-dependent-protein-kinase-pka-catalytic-subunit)</td>
			<td>Peirce PKA is also suitable</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PC, Chicken Egg</td>
			<td>Avanti Polar Lipids (www.avantilipids.com)</td>
			<td>840051C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">POPC</td>
			<td>Avanti Polar Lipids (www.avantilipids.com)</td>
			<td>850457C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PS, Porcine Brain</td>
			<td>Avanti Polar Lipids (www.avantilipids.com)</td>
			<td>840032C</td>
			<td>(CFTR has been successfully reconstituted into Egg PC, POPC, 2:1 (w/w) Egg PC:POPC, or a mixture of PE:PS:PC:ergosterol, 5:2:2:1 (w/w))</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PE, Chicken Egg</td>
			<td>Avanti Polar Lipids (www.avantilipids.com)</td>
			<td>841118C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ergosterol</td>
			<td>Sigma (www.sigmaaldrich.com)</td>
			<td align="right">45480</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pierce Detergent removal spin column</td>
			<td>Thermo Scientific</td>
			<td align="right">87779</td>
			<td>1 ml capacity columns</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">valinomycin</td>
			<td>Sigma (www.sigmaaldrich.com)</td>
			<td>V-0627</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">VX-770 (ivacaftor)</td>
			<td>Selleck Chemicals</td>
			<td>S1144</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">iodide selective microelectrode</td>
			<td>Lazar Research Laboratories (www.shelfscientific.com/cgi-bin/tame/newlaz/microionn.tam)</td>
			<td>LIS-146ICM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Clampex 8.1 software</td>
			<td>Axon Instruments (www.axon.com)</td>
			<td></td>
			<td>we use components of the ClampX system with a home made filter to monitor and record the response to our electrode</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alternate Software: ArrowLabb System&#160;</td>
			<td>Lazar Research Laboratories (www.shelfscientific.com/cgi-bin/tame/newlaz/ionsystems.tam)</td>
			<td>LIS-146LICM-XS</td>
			<td>Lazar Research sells a meter that can interface with a computer and software to record the probe response.&#160; This software should serve a similar function to our setup</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">small stir bars</td>
			<td>Big Science Inc (www.stirbars.com)</td>
			<td>SBM-0502-CMB</td>
			<td>choose a stir bar small enough to easily fit into a well of a 96-well plate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sephadex G50, fine</td>
			<td>GE Health Care (www.gelifesciences.com)</td>
			<td>17-0042-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sonicator</td>
			<td>Laboratory Supplies Co, Inc.</td>
			<td>G112SP1G</td>
			<td>Bath sonicators from other manufacturers should also be suitable</td>
		</tr>
	</tbody>
</table>

functional reconstitution and channel activity measurements of purified wildtype and mutant cftr protein

囊性纤维化跨膜传导调节因子（CFTR）是ATP的一个独特的通道形成构件结合转运盒（ABC）家族。 CFTR的磷酸化和核苷酸依赖的氯离子通道活性已经常研究整个细胞系统和单频道切除膜补丁。许多囊性纤维化致病突变已显示出改变该活动。而少数的纯化方案已经发表，即保留通道活性的快速重建的方法和一种合适的方法来研究人口通道活性的纯化的系统已缺乏。这里迅速的方法描述用于纯化和全长CFTR蛋白的功能重建成保留活性调节的卤化物信道定义的脂质组合物的蛋白脂质体。这个重构方法与通道活性的新颖的磁通为基础的检测一起是一个合适的系统，用于研究的野生型CFTR的人口信道特性和致病突变F508del-和G551D-CFTR。具体地，该方法在研究磷酸，核苷酸和小分子如上CFTR通道活性增强剂和抑制剂的直接影响实用。该方法也适合于其他的膜通道/转运对阴离子基底的研究。

描述这个写入出版物中一些方法来纯化，重建并测量CFTR蛋白的调节的通道活性。 图1A示出的工作流程的纯化，重组和碘化流出程序。方法用于重建和通道活性的测量通过碘化通量也列于进一步的细节，在相关联的视频。 净化CFTR和重建成脂蛋白 CFTR可以功能性以高水平表达于S F使用含有WT或突变体的CFTR基因2重组杆状病毒9细胞。...

Watch this Scientific Journal Video about Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein at JoVE.com

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

这里描述的是一种快速，有效的方法为纯化的野生型和突变型CFTR蛋白的功能性重组，可以保留活动而此氯离子通道，这是有缺陷的囊性纤维化。从由CFTR介导的重组蛋白脂质体碘流出允许通道活性的研究和小分子的影响。

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Research

JoVE Journal

Biology

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

科研

生物学

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Biochemical Reconstitution of Steroid Receptor&#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

类固醇受体的生化重建•Hsp90的蛋白质复合物和配体激活绑定

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, ...

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

（DGN）一Degron不稳定的绿色荧光蛋白（GFP）的报告蛋白在活细胞内泛素 - 蛋白酶体活性的监测

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

赖氨酸去甲基化酶活性的定量测量中的应用的MassSQUIRM

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small ...

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein GST-RhoAG17A from Epithelial Cell Lysates

亲和沉淀主动RHO上皮细胞裂解液中使用的GST标签的突变Rho蛋白（GST-RhoA蛋白（G17A））全环基金

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell ...

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

依赖的PDZ-CFTR的大分子信号复合物的体外分析

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

重组脂膜的结构和功能研究的钾离子通道

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

在植物和绿藻的捕光配合体外重组

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

探讨高密度功能蛋白微阵列来检测蛋白质 - 蛋白质相互作用

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...