Y. Tanaka 对 Y2H 进行了技术改进。这项工作得到了 JSPS KAKENHI Grant Number JP22K06336 和大阪发酵研究所的支持。

1. 突变体库的建设
<ol><li>设置聚合酶链反应 （PCR）（示例如下所示）。通常制备 50 μL 的反应物，将其分成 10 份 5 μL 的等分试样，并在 PCR 仪4 中扩增目标基因片段。 注意:用户应选择适当的聚合酶以有效地引入突变。如果要扩增的DNA片段较短，则需要错误率较高的聚合酶，例如缺乏校对活性的Taq聚合酶。如果要扩增的DNA片段很长，则需要更高保真度的聚合酶来减少突变的数量。使用常规 Taq 聚合酶4 扩增 30 个周期后，每几百个碱基对发生一次突变。在代表性结果中，使用了不同的Taq聚合酶（参见 材料表），其保真度高于常规Taq聚合酶，突变率为600个碱基对中的1。PCR混合物的示例、引物详细信息和反应条件在 补充文件1中提供。</li>
	<li>当PCR完成后，合并反应的所有等分试样，确认目标DNA片段已使用琼脂糖凝胶电泳等法扩增，并纯化DNA5。</li>
	<li>使用"无缝"克隆策略将步骤 1.2 中生成的 DNA 片段与适当的 Y2H 载体组装（图 1）。 注:无缝克隆系统，如Gibson组装6，最大限度地减少 了通过 自连接产生的空载体对构建的突变体文库的污染。 表 1 中提供了 Y2H 载体的列表。它们可以在组装前通过 BamHI 消化来制备。所有质粒均可从国家生物资源项目 - 酵母中获得（见 材料表）。</li>
	<li>将步骤1.3中产生的反应混合物引入根据高效大肠杆菌转化方案制备的大肠杆菌感受态细胞中（例如，Inoue等人7）。将所有感受态细胞铺展在含有适当抗生素的几个LB平板（1%胰蛋白胨，0.5%酵母提取物，1%NaCl和2%琼脂）上（参见材料表）。此时，将少量（总反应的~1/100）涂抹在同一选择板上以计算文库滴度。将板在37°C孵育过夜。</li>
	<li>一旦出现大量 大肠杆 菌菌落，使用一次性塑料环从板表面刮取所有细胞。使用常用方法（如碱性裂解8）从这些细胞中回收质粒 DNA。同时，计算在板上散布转化混合物的小等分试样后出现的菌落数。使用此数字估计库中独立克隆的总数。 注意:此时，选取一些出现在铺有转化反应小等分试样的板上的几个独立菌落（4-6），将它们分开培养，并从中分离质粒，以确认几乎所有克隆都携带所需的DNA片段5。许多商业试剂盒可用于从 大肠杆菌中分离质粒。撒开小等分试样后出现在板上的菌落数量可以手动计算。</li>
	<li>测量文库DNA库的浓度。通常，几百纳克的文库DNA就足以在下一步中获得数千个转化子。 注意:建议使用与 DNA 结合的荧光染料测量 DNA 浓度，因为 A260 的测量经常不准确。</li>
</ol>2. 用突变体文库和复制铺板转化酵母
<ol><li>将突变文库引入宿主酵母菌株（TAT-7（L40-ura3）9,10 MATa，leu2-3,112，trp1-901，his3-Δ200，ade2-101，gal80Δ，LYS2:（lexAop）4-HIS3，ura3:（lexAop）8-lacZ）使用约100ng DNA进行Y2H测定。用"诱饵"质粒预转化宿主细胞。<ol><li>转化程序后，将酵母细胞悬浮在无菌水中，并将不同量的等分试样散布在适当的平板上（通常是缺乏亮氨酸和色氨酸的SC培养基:SC-LW [0.67%酵母氮基，2%葡萄糖，1.546g / L SC双滴混合物-Leu -Trp和2%琼脂，参见 材料表]）。将这些板在30°C下孵育过夜。 注:标准方案，例如醋酸锂-PEG 方法11 ，具有 ~5 × 106 个对数生长的细胞，足以用于酵母的转化。也可以使用另一种转化效率高的方法，例如电穿孔。</li>
	</ol></li>
	<li>第二天，当出现微小的菌落时，选择具有适当细胞数量（500-2000）的板，并按以下步骤从中进行复制。</li>
	<li>将两张滤纸放在复制块上，制成多个复制品（培养基为含有卡那霉素的SC-LW和SC-LW）（见 材料表）。在30°C孵育1-2天。 注:卡那霉素 （G418） 的浓度为 600 μg/mL。不要使用天鹅绒进行复制电镀，因为菌落的分辨率会丢失。</li>
	<li>一旦菌落生长，比较板并选择候选板。如果使用 lacZ 作为报告基因，则执行Y2H颜色测定（参见步骤3）。 注意:对于 Y2H 报告基因， lacZ 通常比 HIS3 更擅长检测弱相互作用。</li>
</ol>3. Y2H显色法
<ol><li>将一张预先切成适当尺寸的滤纸放在使用步骤 2 制备的复制板上。注意不要让气泡在滤纸和板之间形成。 注意: 使用 4A 号滤纸或 50 级滤纸（见 材料表）。任何含有卡那霉素的SC-LW或SC-LW板，都是通过复制电镀制成的，可用于颜色测定。</li>
	<li>当板上的滤纸完全湿润时（当滤纸的颜色完全改变时），在上面放几张纸巾，使其与滤纸接触，以去除多余的水分。当纸巾吸收水分并变色时，请取下纸巾。重复此过程两到三次。</li>
	<li>用镊子捡起滤纸的边缘，迅速将其从板上撕下，浸入液氮中，然后冷冻。如果没有液氮，将滤纸放在塑料托盘上，菌落面朝上，然后立即将其放入冰箱（-20°C至-80°C）中完全冷冻。</li>
	<li>取出冷冻的滤纸，将其放在干燥的纸巾上，菌落面朝上解冻。解冻后，立即将滤纸（菌落面朝上）放在另一张滤纸上，该滤纸已放置在塑料托盘或可密封容器上，并用含有X-gal（5-溴-5-氯-3-吲哚基-β-D-半乳糖苷）和2-巯基乙醇9（参见材料表）的Z-缓冲液（60mM Na2HPO 4,40mM NaH2PO4,10mM KCl和1mM MgSO 4，pH 7.0）浸泡).注意不要让气泡在顶部和底部滤纸之间形成。</li>
	<li>盖上装有滤纸的塑料托盘，将其放入密闭容器或塑料袋中。关闭密闭容器或塑料袋，在37°C下孵育，直到出现蓝色。</li>
	<li>当出现蓝色时，将滤纸（菌落面朝上）放在放置在干净塑料托盘上的约 500 μL 终止溶液 （1 M Na2CO3） 上。停止溶液快速浸泡;因此，请立即取下过滤器，将其放在新鲜的纸巾上，菌落面朝上，然后晾干。</li>
	<li>更换纸巾并且过滤器足够干燥后，使用扫描仪或其他设备捕获数据。</li>
</ol>4. 候选克隆的恢复和确认
<ol><li>从复制品的原始板（或未用于颜色测定的复制板）中选择白色和 KanR 候选克隆。 示例如图 1C 所示。通过在含有卡那霉素的SC-LW培养基上将它们重新划线为小斑块，并在30°C下孵育1-2天，确认候选克隆是白色和KanR 。至少制作两组或在第二天进行复制。 注意:应选择白色菌落，因为淡蓝色菌落可能会保留相互作用。</li>
	<li>一旦候选克隆重新生长良好，如步骤3中所述，使用步骤4.1中生成的至少一个板重复颜色测定，并确认它们保持白色并且不会变成蓝色（图2A）。 注意:重新划线候选细胞时，不要携带大量细胞。细胞过多导致无法区分 KanR 和 KanS 克隆。</li>
	<li>从板残渣中取出仍然是白色和步骤4.2中产生的KanR 的克隆，并在1mL选择培养基（SC-L或SC-W，取决于用于文库构建的载体）中独立生长过夜。</li>
	<li>将培养物转移到微管中并通过离心（~15,000 × g，在室温下 10-30 秒）收集细胞。按如下方式从细胞沉淀中分离全 DNA。</li>
	<li>将细胞沉淀悬浮在含有1μL2-巯基乙醇和20mg / mL酶解酶100T的400μL裂解缓冲液（2%Triton X-100,1%SDS，100mM NaCl，10mM Tris-Cl（8.0）和1mM EDTA中（参见 材料表），并在37°C孵育30分钟以消化细胞壁。此时，每 5-10 分钟倒置试管轻轻混合。</li>
	<li>当细胞悬液变得不透明或半透明时，加入等体积的苯酚 - 氯仿 - 异戊醇（25:24:1）混合物，并通过中等速度涡旋10-20秒充分混合。</li>
	<li>在微量离心机中以全速（~15,000× g，室温）离心5分钟，并在新的微管中回收含有DNA的水相。加入 0.8 体积的异丙醇，倒置试管充分混合。</li>
	<li>将试管在室温下放置 2-3 分钟，然后在微量离心机中以全速（~15,000 × g，室温）离心 4-5 分钟以沉淀 DNA。</li>
	<li>除去上清液，用约500μL的70%乙醇洗涤沉淀物。完全去除乙醇并短暂干燥沉淀物。</li>
	<li>向沉淀物中加入 20 μL TE 缓冲液（10 mM Tris-Cl （8.0） 和 1 mM EDTA），短暂混合，并在室温下离开试管过夜以溶解沉淀物。 注意:如果用户赶时间，请将试管保持在 37-50 °C。 DNA 沉淀物将在几个小时内溶解。</li>
	<li>一旦沉淀完全溶解，用少量的这种DNA溶液转化 大肠杆菌 。 注:如果使用转化效率高的大 肠杆菌 ，则约 0.5 μL DNA 溶液就足够了。</li>
	<li>当 出现大肠杆菌 菌落时，选择几个独立的菌落，培养它们，并通过标准方法（例如碱性裂解）回收质粒。</li>
	<li>将步骤4.12中获得的质粒DNA引入含有诱饵质粒的Y2H宿主细胞中。当转化体出现时，通过颜色测定法（它们应显示为白色）和蛋白质印迹4 （它们应表达所需大小的蛋白质）进行检查。 注:后碱性方法12 是从酵母制备全细胞提取物的一种简单快速的方法。</li>
	<li>如果满足上述两个标准，则通过核苷酸测序4确定克隆的突变位点。</li>
</ol>

使用酵母双杂交技术的生物分子相互作用研究 

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本文介绍如何使用 Y2H 检测分离相互作用无效/受损的突变体。这种突变体是分析目标蛋白质功能的有力工具。为了分离此类突变体，以前通过修饰 Y2H 宿主菌株 2,3 开发了 rY2H 测定法。然而，他们并没有大大减少劳动量。相比之下，可以使用这种方法分离突变体，而无需大量的劳动力。在这种方法中，理论上，对Y2H载体的修饰可确保所有候选突变体都是?...

生物分子之间的相互作用是生物现象中最基本的部分。蛋白质-蛋白质相互作用构成了这种相互作用的重要组成部分。因此，鉴定目标蛋白的相互作用伙伴对于进一步阐明目标蛋白质/基因的功能至关重要。酵母双杂交 （Y2H） 方法是一种用于鉴定体内蛋白质-蛋白质相互作用的常用技术 1。在该系统中，要测试其相互作用的两种蛋白质（X 和 Y）分别融合到 DNA 结合 （DB） 结构域和转录激活结构域 （AD）。DB-X 融合蛋白与 DB 结构域的识别序列结合;因此，当蛋白质 X 和 Y 相互作用时，AD-Y 融合蛋白就会接近识别序列。因此，识别序列下游的报告基因转录被激活。因此，报告基因活性的存在与否可用于确定蛋白质-蛋白质相互作用的存在与否1。
一旦确定了目标蛋白质的特定相互作用伙伴，就应进行进一步分析以阐明相互作用的生物学功能。为此，如果可以分离出损害或去除特定蛋白质-蛋白质相互作用的蛋白质突变体，它们将成为强大的工具。Y2H系统可以通过筛选"相互作用阴性"克隆直接用于分离此类突变体，从野生型"相互作用阳性"克隆开始。为了加速这一过程，开发了"反向"Y2H （rY2H） 系统 2,3。在 rY2H 系统中，宿主酵母菌株具有反选标记基因作为报告基因，这意味着酵母细胞仅在 AD-Y 和 DB-X 蛋白不相互作用时生长。
尽管 Y2H 和 rY2H 系统都允许分离相互作用阴性突变体，但分离突变体的过程非常费力，因为并非所有通过筛选获得的候选基因都携带所需类型的突变（通常是错义突变）。最严重的问题是，很大一部分候选基因存在移码或无义突变，因此有必要进行蛋白质印迹法以排除不需要的克隆。为了克服这个问题，已经开发了新的质粒载体4.在这些向量中，KanMX（一种耐药性标记物）位于 DB 域或 AD 下游的帧外。只有当目的基因入时，标记基因才会与DB结构域或AD一起进入框架。当在目的基因中引入随机突变时，不需要的突变体，例如具有移码或无义突变的突变体，可以通过进行耐药性选择轻松消除，并且携带理想错义突变的候选突变可以通过Y2H筛选轻松识别4。本文提出了一种方案，用于使用该策略分离目标蛋白质的相互作用无效/受损突变体。

<table><tbody><tr><td>0.5 M EDTA (8.0)</td><td>Nacalai Tesque Inc.</td><td>14347-21</td><td></td></tr><tr><td>10% SDS Solution</td><td>Fujifilm Wako Pure Chemical Corp.</td><td>313-90275</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Fujifilm Wako Pure Chemical Corp.</td><td>135-07522</td><td></td></tr><tr><td>2-propanol</td><td>Kishida Chemical Co. Ltd.</td><td>110-64785</td><td></td></tr><tr><td>5-Bromo-4-chloro-3-indolyl-&amp;beta;-D-galactopyranoside (X-Gal)</td><td>Fujifilm Wako Pure Chemical Corp.</td><td>021-07852</td><td></td></tr><tr><td>Agar</td><td>Formedium</td><td>AGR60</td><td></td></tr><tr><td>Ampicillin Sodium</td><td>Fujifilm Wako Pure Chemical Corp.</td><td>68-52-3</td><td></td></tr><tr><td>Anti-HA-tag mAb-HRP-DirecT&amp;nbsp;&amp;nbsp;</td><td>Medical &amp;amp; Biological Laboratories Co. Ltd.</td><td>M180-7</td><td></td></tr><tr><td>DNA from salmon testes</td><td>Merck KGaA.</td><td>D1626</td><td></td></tr><tr><td>Ethanol</td><td>Merck KGaA.</td><td>9-0770-4-4L-J</td><td></td></tr><tr><td>Filter paper for colony lift (Grade 50)</td><td>Whatman, Cytiva</td><td>1450-090</td><td></td></tr><tr><td>Filter paper for colony lift (No.4A)</td><td>Advantec Toyo Kaisha, Ltd.</td><td>01411090</td><td></td></tr><tr><td>Filter paper for replicaplating (No.1)</td><td>Advantec Toyo Kaisha, Ltd.</td><td>00011150</td><td></td></tr><tr><td>G-418 Sulfate</td><td>Fujifilm Wako Pure Chemical Corp.</td><td>075-05962</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Kishida Chemical Co. Ltd.</td><td>230-37585</td><td></td></tr><tr><td>KCl</td><td>Fujifilm Wako Pure Chemical Corp.</td><td>163-03545</td><td></td></tr><tr><td>Lithium Acetate Dihydrate</td><td>Nacalai Tesque Inc.</td><td>20604-22</td><td></td></tr><tr><td>MgSO4&amp;bull;7H2O</td><td>Fujifilm Wako Pure Chemical Corp.</td><td>131-00405</td><td></td></tr><tr><td>Na2HPO4&amp;bull;12H2O</td><td>Nacalai Tesque Inc.</td><td>10039-32-4</td><td></td></tr><tr><td>NaCl</td><td>Nacalai Tesque Inc.</td><td>31319-45</td><td></td></tr><tr><td>NaH2PO4&amp;bull;2H2O</td><td>Nacalai Tesque Inc.</td><td>31717-25</td><td></td></tr><tr><td>Paper towel</td><td>AS ONE Corp.</td><td>7-6200-02</td><td></td></tr><tr><td>Phenol:Chloroform:Isoamyl Alcohol 25:24:1</td><td>Nacalai Tesque Inc.</td><td>25970-56</td><td></td></tr><tr><td>Plasmid DNAs</td><td>the National BioResource Project - yeast (https://yeast.nig.ac.jp/yeast/top.xhtml)</td><td></td><td></td></tr><tr><td>Plasmid isolation Kit</td><td>Nippon Genetics Co. Ltd.</td><td>FG-90502</td><td></td></tr><tr><td>Polyethylene Glycol #4,000</td><td>Nacalai Tesque Inc.</td><td>11574-15</td><td></td></tr><tr><td>SC double drop-out mix -Leu -Trp</td><td>Formedium</td><td>DSCK172</td><td></td></tr><tr><td>Seamless cloning kit (In-Fusion assembly )</td><td>Takara Bio Inc.</td><td>#639648</td><td></td></tr><tr><td>Skim milk powder</td><td>Fujifilm Wako Pure Chemical Corp.</td><td>190-12865</td><td></td></tr><tr><td>Streptomycin Sulfate</td><td>Fujifilm Wako Pure Chemical Corp.</td><td>3810-74-0</td><td></td></tr><tr><td>Taq polymerase (GoTaq Green Master Mixes)</td><td>Promega Corp.</td><td>M7122</td><td></td></tr><tr><td>TRIS (hydroxymethyl) aminomethane</td><td>Formedium</td><td>TRIS01</td><td></td></tr><tr><td>Triton X-100</td><td>Nacalai Tesque Inc.</td><td>12967-45</td><td></td></tr><tr><td>Tryptone</td><td>ThermoFisher scientific Inc.</td><td>211705</td><td></td></tr><tr><td>Tween 20</td><td>Nacalai Tesque Inc.</td><td>35624-15</td><td></td></tr><tr><td>Yeast Extract</td><td>ThermoFisher scientific Inc.</td><td>212750</td><td></td></tr><tr><td>Yeast Nitrogen Base (YNB)</td><td>Formedium</td><td>CYN0210</td><td></td></tr><tr><td>Zymolyase 100T</td><td>Nacalai Tesque Inc.</td><td>07665-55</td><td></td></tr></tbody></table>

isolating interaction-null&#47;impaired mutants using the yeast two-hybrid assay

蛋白质-蛋白质相互作用是构成生物现象的最基本过程之一。理解特定蛋白质-蛋白质相互作用的作用和功能的最简单和最好的方法之一是比较野生型的表型（具有相关的蛋白质-蛋白质相互作用）和缺乏相关相互作用的突变体的表型。因此，如果能够分离出这样的突变体，它们将有助于阐明相关的生物学过程。酵母双杂交 （Y2H） 程序是一种强大的方法，不仅可以检测蛋白质-蛋白质相互作用，还可以分离相互作用无效/受损的突变体。在本文中，提出了一种使用 Y2H 技术分离相互作用无效/受损突变体的协议。首先，将聚合酶链反应与高效的无缝克隆技术相结合，构建突变文库，有效地从文库中排除空载体;其次，通过Y2H测定筛选相互作用无效/受损的突变体。由于 Y2H 载体中的一个技巧，不需要的突变体，例如具有移码和无义突变的突变体，可以有效地从筛选过程中消除。这种策略很简单，因此可以应用于任何蛋白质组合，其相互作用可以被双杂交系统检测到。

最近，发现Pol2蛋白（Pol2-C）的C端半部分与Mcm10相互作用。这两种蛋白质对于启动 DNA 复制都是必需的，因此对于出芽酵母酿酒酵母 13,14,15 中的细胞生长都是必需的。为了帮助理解这种相互作用的生物学意义，使用此处描述的方法分离出与 Mcm10 没有/减少相互作用的 Pol2-C 突变体。
按照步骤1中描述的?...

Watch this Scientific Journal Video about Author Spotlight: Enhanced Isolation of Interaction-Null Mutants in Yeast at JoVE.com

作者聚焦:增强酵母中相互作用零突变体的分离

生物分子的相互作用，如蛋白质-蛋白质相互作用，是生物学功能的分子基础。如果可以分离出特别缺乏相关相互作用的相互作用无效/受损突变体，它们将极大地有助于理解这种相互作用的功能。本文提出了一种有效的方法来分离相互作用无效/受损的突变体。

使用酵母双杂交试验分离相互作用无效/受损突变体

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the ...

我们对了解真核生物细胞周期的调控机制感兴趣，特别是DNA复制、稳定胚芽维持和细胞增殖之间的关系。真核生物中细胞周期的进展受细胞周期蛋白依赖性激酶（CDK）的调节。DNA复制的启动就是这种情况。我们已经在CDK的底物上发现了一种启动DNA复制的方法。要了解您感兴趣的蛋白质的功能，比较野生型和突变体是一种直接而有效的方法。酵母双杂交试验，是简单的检测方法，将增强分离相互作用无效突变体的过程。这将有助于推动该领域的研究。

isolating-interaction-nullimpaired-mutants-using-yeast-two-hybrid

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Isolating Interaction-Null&#47;Impaired Mutants Using the Yeast Two-Hybrid Assay

347 次观看.  Kochi University of Technology. 我们对了解真核生物细胞周期的调控机制感兴趣，特别是DNA复制、稳定胚芽维持和细胞增殖之间的关系。真核生物中细胞周期的进展受细胞周期蛋白依赖性激酶（CDK）的调节。DNA复制的启动就是这种情况。我们已经在CDK的底物上发现了一种启动DNA复制的方法。要了解您感兴趣的蛋白质的功能，比较野生型和突变体是一种直接而有效的方法。酵母双杂交试验，是简单的检?...

视频: 作者聚焦:增强酵母中相互作用零突变体的分离

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the discovery of high-affinity interactors that are highly enriched in the library of interacting candidates. In a traditional format, the yeast 2-hybrid assay can yield too many colonies to analyze when conducted at low stringency where low affinity interactors might be found. Moreover, without a comprehensive and complete interrogation of the same library against different bait plasmids, a comparative analysis cannot be achieved. Although some of these problems can be addressed using arrayed prey libraries, the cost and infrastructure required to operate such screens can be prohibitive. As an alternative, we have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing a strategy termed DEEPN (Dynamic Enrichment for Evaluation of Protein Networks), which incorporates high-throughput DNA sequencing and computation to follow the evolution of a population of plasmids that encode interacting partners. Here, we describe customized reagents and protocols that allow a DEEPN screen to be executed easily and cost-effectively.

Batch processing of yeast 2-hybrid screens allows for direct comparison of the interaction profiles of multiple bait proteins with a highly complex set of prey fusion proteins. Here, we describe refined methods, new reagents, and how to implement their use for such screens.

酵母 2-混合筛在批次中比较蛋白质相互作用

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

科研

生物化学

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

一种在裂变酵母中的深度固序辅助自发抑制屏

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

遗传学

Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated biological pathways to the benefit of the bacterium. Identification of putative bacterial effector proteins has become more manageable due to advances in bacterial genome sequencing and the advent of algorithms that allow in silico identification of genes encoding secretion candidates and/or eukaryotic-like domains. However, identification of these important virulence factors is only an initial step. Naturally, the goal is to determine the molecular function of effector proteins and elucidate how they interact with the host. In recent years, techniques like the yeast two-hybrid screen and large-scale immunoprecipitations coupled with mass spectrometry have aided in the identification of protein-protein interactions. Although identification of a host binding partner is the crucial first step toward elucidating the molecular function of a bacterial effector protein, sometimes the host protein is found to have multiple biological functions (e.g., actin, clathrin, tubulin), or the bacterial protein may not physically bind host proteins, depriving the researcher of crucial information about the precise host pathway being manipulated. A modified yeast toxicity screen coupled with a suppressor screen has been adapted to identify host pathways impacted by bacterial effector proteins. The toxicity screen relies on a toxic effect in yeast caused by the effector protein interfering with the host biological pathways, which often manifests as a growth defect. Expression of a yeast genomic library is used to identify host factors that suppress the toxicity of the bacterial effector protein and thus identify proteins in the pathway that the effector protein targets. This protocol contains detailed instructions for both the toxicity and suppressor screens. These techniques can be performed in any lab capable of molecular cloning and cultivation of yeast and Escherichia coli.

Bacterial pathogens secrete proteins into the host that target crucial biological processes. Identifying the host pathways targeted by bacterial effector proteins is key to addressing molecular pathogenesis. Here, a method using a modified yeast suppressor and toxicity screen to elucidate host pathways targeted by toxic bacterial effector proteins is described.

使用酵母毒性和抑制器屏幕识别细菌效应蛋白的宿主通路

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

基于分体式泛素膜酵母双杂交（神话）系统：一个强大的工具用于识别蛋白质 - 蛋白质相互作用

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

生物学

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

从非耕种植物病原菌利用酵母双杂交筛选Unravelling一个细菌效应中的作用

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

免疫与感染

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

改良酵母双杂交系统，以确定蛋白与生长因子Progranulin的相互作用

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

反向酵母双杂交系统，以确定哺乳动物核受体残留的互动与配体和/或拮抗剂

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic lateral sclerosis, and &#945;-synuclein, implicated in Parkinson&#8217;s disease, are toxic and form cytoplasmic aggregates in yeast. These features recapitulate protein pathologies observed in patients with these disorders. Thus, yeast are an ideal platform for isolating toxicity suppressors from libraries of protein variants. We are interested in applying protein disaggregases to eliminate misfolded toxic protein conformers. Specifically, we are engineering Hsp104, a hexameric AAA+ protein from yeast that is uniquely capable of solubilizing both disordered aggregates and amyloid and returning the proteins to their native conformations. While Hsp104 is highly conserved in eukaryotes and eubacteria, it has no known metazoan homologue. Hsp104 has only limited ability to eliminate disordered aggregates and amyloid fibers implicated in human disease. Thus, we aim to engineer Hsp104 variants to reverse the protein misfolding implicated in neurodegenerative disorders. We have developed methods to screen large libraries of Hsp104 variants for suppression of proteotoxicity in yeast. As yeast are prone to spontaneous nonspecific suppression of toxicity, a two-step screening process has been developed to eliminate false positives. Using these methods, we have identified a series of potentiated Hsp104 variants that potently suppress the toxicity and aggregation of TDP-43, FUS, and &#945;-synuclein. Here, we describe this optimized protocol, which could be adapted to screen libraries constructed using any protein backbone for suppression of toxicity of any protein that is toxic in yeast.

Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.

隔离增效变种的Hsp104利用酵母Proteinopathy模型

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

遗传和生物化学方法<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;蛋白齐聚评估:兰尼碱受体案例研究

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

Modified Yeast-One Hybrid Assay to Detect Heteromeric Protein Complex-DNA Interactions

Source: Tripathi, P. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/56080">A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies.</a> J. Vis. Exp. (2017)
In this video, we demonstrate the modified yeast one-hybrid assay, which detects multiple proteins interacting with target DNA. The transformed yeast cells contain the target DNA sequence upstream of the reporter gene. Additionally, they express transcription factor proteins. When the proteins form complexes and bind to the target DNA, the reporter gene is expressed and can be detected enzymatically.

用于检测异聚蛋白复合物-DNA 相互作用的改良酵母-One 杂交检测

Source: Tripathi, P. et al., A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies. J. Vis. Exp. (2017)
In this ...