Name: pulldown assay coupled with co-expression in bacteria cells as a time-efficient tool for testing challenging protein-protein interactions
Uploaded: 2022-12-23T14:00:00
Description: Watch this Scientific Journal Video about Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions at JoVE.com

这项工作得到了俄罗斯科学基金会项目19-74-30026（方法开发和验证）和19-74-10099（蛋白质-蛋白质相互作用测定）的支持;以及俄罗斯联邦科学和高等教育部授予075-15-2019-1661（代表性蛋白质 - 蛋白质相互作用的分析）。

方法工作流程的示意图如图 1所示。
1. 大肠杆菌的共转化
<ol><li>使用标准克隆方法制备靶蛋白的表达载体。 注意：通常，一个好的起点是使用带有氨苄青霉素抗性基因和ColE1来源的常规pGEX/pMAL载体表达GST/MBP标记蛋白，以及使用具有p15A或RSF来源和卡那霉素抗性的兼容载体来表达6xHis标记的蛋白质，在某些情况下与硫氧还蛋白或SUMO标...

<ol>
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所描述的方法允许测试不能在 体外 有效组装并需要共表达的蛋白质 - 蛋白质相互作用。该方法是研究异源二聚化蛋白质的少数合适方法之一，这些蛋白质也能够进行同源二聚化，因为当单独纯化时，这些蛋白质形成稳定的同源二聚体，在实验3，11期间通常不能解离和重新结合。
与传统的下拉相比，所述方法的工作流程更具?...

常规下拉广泛用于研究蛋白质-蛋白质相互作用1。然而，纯化的蛋白质通常不能在体外有效相互作用2，3，其中一些在没有结合伴侣4，5的情况下是不溶的。这些蛋白质可能需要共翻译组装或分子伴侣5，6，7，8，9的存在。常规下拉的另一个限制是测试结构域之间可能的异多聚化活性，这些结构域可以作为稳定的同源低聚物以共翻译8，10的形式存在，因为它们中的许多在孵育期间不能在体外解离和重新结合。发现共表达有助于克服这些问题3，11。使用细菌中相容载体的共表达成功用于纯化大型多亚基大分子复合物，包括Polycomb抑制复合物PRC2 12，RNA聚合酶II介质头模块13，噬菌体T4底板14，SAGA复合物去泛素化酶模块15，16和铁蛋白17。通常用于共表达的复制起源是ColE1，p15A18，CloDF1319和RSF20。在市售的Duet表达系统中，这些来源与不同的抗生素抗性基因和方便的多个克隆位点相结合，产生多顺反子载体，允许表达多达八种蛋白质。这些来源具有不同的拷贝数，可以以不同的组合使用，以实现靶蛋白的平衡表达水平21。为了测试蛋白质-蛋白质相互作用，使用了各种亲和标签;最常见的是6x组氨酸，谷胱甘肽-S-转移酶（GST）和麦芽糖结合蛋白（MBP），每种都对相应的树脂具有特定的亲和力。GST和MBP还增强了标记蛋白的溶解度和稳定性22。
还开发了许多涉及真核细胞中蛋白质共表达的方法，其中最突出的是酵母双杂交测定（Y2H）23。Y2H测定便宜，简单，并且允许测试多种相互作用;但是，其工作流程需要 1 周以上才能完成。还有一些不太常用的基于哺乳动物细胞的测定，例如荧光双杂交测定（F2H）24 和细胞阵列蛋白质 - 蛋白质相互作用测定（CAPPIA）25。F2H测定相对较快，允许观察其天然细胞环境中的蛋白质相互作用，但涉及使用昂贵的成像设备。所有这些方法都比原核表达具有优势，可提供天然真核翻译和折叠环境;然而，它们通过转录激活或荧光能量转移间接检测相互作用，这通常会产生伪影。此外，真核细胞可能包含目标蛋白质的其他相互作用伙伴，这可以干扰高等真核生物蛋白质之间二元相互作用的测试。
本研究描述了一种通过常规下拉技术对差异标记蛋白进行细菌共表达的方法。该方法允许研究需要共表达的靶蛋白之间的相互作用。与传统的下拉相比，它更省时，允许对多个目标进行批量测试，这在大多数情况下具有优势。使用兼容载体的共表达比多顺反子共表达更方便，因为它不需要费力的克隆步骤。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>8-ELEMENT probe</td>
			<td>Sonics</td>
			<td>630-0586</td>
			<td>The high throughput 8-element sonicator probes</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>AppliChem</td>
			<td>A0949</td>
			<td></td>
		</tr>
		<tr>
			<td>Amylose resin</td>
			<td>New England Biolabs</td>
			<td>E8021</td>
			<td>Resin for purification of MBP-tagged proteins</td>
		</tr>
		<tr>
			<td>Antibiotics</td>
			<td>AppliChem</td>
			<td>A4789 (kanamycin); A0839 (ampicillin)</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta-mercaptoethanol</td>
			<td>AppliChem</td>
			<td>A1108</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21(DE3)&#160;</td>
			<td>Novagen</td>
			<td>69450-M</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>AppliChem</td>
			<td>A4689</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5415R (Z605212)</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione</td>
			<td>AppliChem</td>
			<td>A9782</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione agarose</td>
			<td>Pierce</td>
			<td>16100</td>
			<td>Resin for purification of GST-tagged proteins</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>AppliChem</td>
			<td>A2926</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES&#160;</td>
			<td>AppliChem</td>
			<td>A3724</td>
			<td></td>
		</tr>
		<tr>
			<td>HisPur Ni-NTA Superflow Agarose</td>
			<td>Thermo Scientific</td>
			<td>25214</td>
			<td>Resin for purification of 6xHis-tagged proteins</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>AppliChem</td>
			<td>A1378</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>AppliChem</td>
			<td>A4773</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>AppliChem</td>
			<td>A2939</td>
			<td></td>
		</tr>
		<tr>
			<td>LB</td>
			<td>AppliChem</td>
			<td>414753</td>
			<td></td>
		</tr>
		<tr>
			<td>Maltose</td>
			<td>AppliChem</td>
			<td>A3891</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>AppliChem</td>
			<td>A2947</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>AppliChem</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr>
			<td>NP40</td>
			<td>Roche</td>
			<td>11754599001</td>
			<td></td>
		</tr>
		<tr>
			<td>pACYCDuet-1</td>
			<td>Sigma-Aldrich</td>
			<td>71147</td>
			<td>Vector for co-expression of proteins with p15A replication origin</td>
		</tr>
		<tr>
			<td>pCDFDuet-1</td>
			<td>Sigma-Aldrich</td>
			<td>71340</td>
			<td>Vector for co-expression of proteins with CloDF13 replication origin</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>AppliChem</td>
			<td>A0999</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail VII</td>
			<td>Calbiochem</td>
			<td>539138</td>
			<td>Protease Inhibitor Cocktail</td>
		</tr>
		<tr>
			<td>pRSFDuet-1</td>
			<td>Sigma-Aldrich</td>
			<td>71341</td>
			<td>Vector for co-expression of proteins with RSF replication origin</td>
		</tr>
		<tr>
			<td>SDS&#160;</td>
			<td>AppliChem</td>
			<td>A2263</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris&#160;</td>
			<td>AppliChem</td>
			<td>A2264</td>
			<td></td>
		</tr>
		<tr>
			<td>VC505 sonicator</td>
			<td>Sonics</td>
			<td>CV334</td>
			<td>Ultrasonic liquid processor</td>
		</tr>
		<tr>
			<td>ZnCl2</td>
			<td>AppliChem</td>
			<td>A6285</td>
			<td></td>
		</tr>
	</tbody>
</table>

pulldown assay coupled with co-expression in bacteria cells as a time-efficient tool for testing challenging protein-protein interactions

下拉是一种简单且广泛使用的蛋白质-蛋白质相互作用测定。然而，它在研究体 外不能有效组装的蛋白质复合物方面存在局限性。这种复合物可能需要共翻译组装和分子伴侣的存在;它们要么形成稳定的低聚物，在 体外 不能解离和重新结合，要么在没有结合伴侣的情况下不稳定。为了克服这些问题，可以使用基于差异标记蛋白的细菌共表达的方法，使用一组兼容的载体，然后使用传统的下拉技术。与传统的下拉相比，该工作流程更省时，因为它缺乏单独纯化相互作用蛋白及其后续孵育的耗时步骤。另一个优点是更高的可重复性，因为 体外 环境中存在的蛋白质暴露于蛋白水解和氧化的步骤数量明显较少，并且持续时间更短。当发现其他 体外 技术不合适时，该方法成功地应用于研究许多蛋白质 - 蛋白质相互作用。该方法可用于批量测试蛋白质-蛋白质相互作用。对于BTB结构域与固有无序蛋白质之间的相互作用以及锌指相关结构域的异二聚体的研究，显示了具有代表性的结果。

所描述的方法常规用于许多不同的目标。这里介绍的是一些使用传统下拉技术可能无法获得的代表性结果。首先是研究特异性ZAD（锌指相关结构域）二聚化11。ZAD形成稳定和特异性的二聚体，异二聚体只能在副同源组内密切相关的结构域之间产生。由这些结构域形成的二聚体是稳定的，至少几天内不会解离;因此，混合纯化的ZAD不会产生任何可检测的结合。同时，MBP和硫氧还蛋白-...

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Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

在这里，我们描述了一种使用一组兼容载体对差异标记蛋白质进行细菌共表达的方法，然后使用传统的下拉技术来研究无法 在体外组装的蛋白质复合物。

下拉测定与细菌细胞共表达相结合，作为测试具有挑战性的蛋白质-蛋白质相互作用的省时工具

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble ...

该方法允许研究需要共表达的蛋白质复合物的形成，以实现有效的测试样品，例如异二聚体的形成。该方法的主要最终潜力是利用差异标记研究蛋白质的共表达，使用兼容载体的组合来促进高效的复杂组装，以及节省时间的工作流程和可扩展性，允许对多种相互作用进行快速斑贴测试。该方法还可用于快速筛选最佳蛋白表达和纯化条件。可视化演示有助于正确执行实验方案的关键步骤，例如共表达设置、细胞破碎和后续下拉步骤。首先，在 37 摄氏度的 Luria-Bertani（LB）培养基中培养大肠杆菌 BL21（DE3）菌株，使其光密度或 OD 为 0.1 至 0.2。在4摄氏度下以9， 000g离心一毫升细菌悬浮液一分钟，弃去上清液。然后，加入0.5毫升冰冷的转化缓冲液或TB，并在冰上孵育10分钟。孵育结束时，在4摄氏度下以8， 000G离心30秒，弃去上清液。然后，加入100微升TB缓冲液，每种载体100纳克，并在冰上孵育30分钟。在 42 摄氏度下加热 150 秒，然后在冰上冷却一分钟。接下来，加入一毫升不含抗生素的LB培养基，并在37摄氏度下孵育90分钟。孵育后，将沉淀离心并重悬于100微升LB培养基中，然后将悬浮液接种在含有0.5%葡萄糖和相应抗生素的LB琼脂平板上。将板在 37 摄氏度下孵育过夜。第二天，用两毫升LB培养基将平板上的细胞冲洗到含有相应抗生素的50毫升LB培养基中。在将含有20%甘油的等分试样储存在零下70摄氏度之前，加入金属离子或其他已知的辅助因子，以进行后续实验。在 220 摄氏度下以 37 RPM 的恒定旋转培养细胞，以 0.5 至 0.7 的 OD 培养细胞。将培养物冷却至室温，并加入IPTG至终浓度为1毫摩尔。储存20微升等分试样的细胞悬液作为未诱导样品的对照。将细胞在 220 RPM 和 18 摄氏度下孵育过夜。第二天，将细菌悬浮液分成两部分并储存20微升等分试样以确认蛋白质表达。将剩余的细胞悬液以4， 000 G离心15分钟。对于下拉测定，将沉淀重悬于一毫升冰冷的裂解缓冲液中，并在实验前立即加入蛋白酶抑制剂和还原剂。调整测试蛋白质的缓冲液组成。通过在冰上超声处理破坏细胞，并储存20微升等分试样用于电泳。以16， 000G离心30分钟，收集20微升澄清裂解物用于电泳。用一毫升冰冷的裂解缓冲液平衡树脂10分钟，然后以2， 000g离心30秒并弃去上清液。将细胞裂解物添加到树脂中，并以15RPM的恒定旋转孵育10分钟。然后，离心并弃去上清液，然后收集20微升未结合的馏分进行分析。接下来，加入一毫升冰冷的洗涤缓冲液并孵育一分钟。再次，离心并丢弃上清液。然后，用冰冷的洗涤缓冲液进行两次长时间洗涤。第二次洗涤后，用50微升洗脱缓冲液在摇床中以1， 200 RPM在4摄氏度下洗脱结合的蛋白质10分钟。用SDS聚丙烯酰胺凝胶电泳分析洗脱的蛋白质。本文显示了锌指相关结构域（ZAD）、麦芽糖结合蛋白（MBP）中的同源二聚化和6x组氨酸下拉测定的研究。MBP和硫氧还蛋白6x组氨酸融合ZADs的共表达显示出良好且可重现的同源二聚化活性，在MBP下拉测定的SDS-PAGE结果中显示为附加条带。这里显示了使用该方法研究替代复合物形成的另一个示例。在共表达测定中，6x组氨酸标记的ENY2与GST标记的Sgf11和MBP-CTCF相互作用，但在MBP下拉中不存在GST-Sgf11，反之亦然。此处显示了传统下拉测定工作流程中所需时间间隔与下拉与共表达耦合的分步比较。此处显示了利用这两种技术研究CP190蛋白的BTB结构域与果蝇CTCF蛋白的GST标记C末端结构域之间相同相互作用的结果。正确选择亲和力和溶解度测试、硫氧还蛋白、GST 或 MBP 对于有效执行测定至关重要。另一个关键步骤是快速均匀的细胞破碎。强烈建议使用多尖端超声探头。该方法允许直接研究蛋白质结构域之间的异二聚体形成，否则形成在常规下拉测定中纯化蛋白质孵育期间不会解离的同源二聚体。

Research

JoVE Journal

Biochemistry

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

荧光各向异性作为一种工具来研究蛋白质相互作用

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

科研

生物化学

Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria

利用乙烯释放化合物，2-氯乙基膦酸，作为一种工具来研究细菌乙烯反应

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

化细胞穿透肽研究胞内蛋白-蛋白质相互作用

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

Variations on Negative Stain Electron Microscopy Methods: Tools for Tackling Challenging Systems

Negative stain electron microscopy (EM) allows relatively simple and quick observation of macromolecules and macromolecular complexes through the use of ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

生物层干涉测量(BLI)在转录中研究蛋白质-蛋白质相互作用的应用

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

淀粉样蛋白纤维蛋白的相互作用和脑线粒体的膜渗透

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria

MS2亲和力纯化与革兰阳性细菌中的RNA测序相结合

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized ...