这项工作是由卫生研究院（NIH）国家机构支持的补助CA127231及达蒙鲁尼恩基金会临床研究奖（CI 1502）（以SM）。我们要感谢的Zdenek德沃夏克教授帕拉茨基大学奥洛穆茨，捷克共和国的有益见解讨论这个技术移植到他们的机构和协议的标准化。

1。 PXR和SRC-1融合体在酵母载体的构建<ol><li> PCR扩增人类PXR LBD（107-434氨基酸）和人类SRC-1全长（1-1401氨基酸）。 <ol><li>使用pSG5-hPXR 8质粒作为PXR LBD模板，使用PCMX-SRC1质粒为SRC-1的模板。 </li><li>解冻PCR SuperMix，DNA模板和引物（见材料），让他们在冰上。 </li><li>加0.25微克/微升DNA模板1微升，10毫引物对2微升各自的PCR SuperMix 45微升，并添加H 2 O至使总体积为50微升。 </li><li>设置PCR在94℃下一个循环2分钟，20个循环的94℃30秒，55℃30秒，72℃2分钟，然后72℃10分钟延长一个周期。检查PCR产物用1％琼脂糖凝胶电泳。 </li></ol></li><li> PCR产物克隆到酵母双杂交载体。 <ol><li>从1％琼脂糖凝胶纯化的PCR产物。摘要PXR，LBD，PCR和Y2H载体pSH2-1BamHI和SalI位;消化SRC-1 PCR和Y2H载体pGADNOT与NotΙ和SalΙ。要做到这一点，加1微克的DNA，3微升10X反应缓冲液，1μL限制性内切酶，最后H 2 O至使总体积为30μL。 </li><li>把消解样品在37℃水浴1小时。通过在1％琼脂糖凝胶电泳检查消化。 </li><li>纯化消化的样品在1％琼脂糖凝胶上，结扎消化的PCR产物和Y2H载体和转化到DH5α感受态细胞。 </li><li>挑殖民地和隔离质粒DNA。 </li></ol></li><li>识别pSH2-1-PXR质粒DNA经BamHI / SalI位消化，找出pGADNOT-SRC-1质粒DNA用NotI位/ SalI位消化。通过测序验证一切积极质粒DNA。 </li></ol> 2。酵母双杂交试验<ol><li>接种酵母菌株到5ml YPAD孵育过夜通过不断搅拌（220转，30℃）。注意: 酿酒酵母的菌株是CTY10-5D（MAT一个ADE2 TRP 1-901 LEU2-3，112 HIS3-200 GA L4 - GAL80 - URA3 :: LEXA-lacZ的 ），其中包含一个集成的GAL1-lacZ基因与LexA操纵 10。要获得可行的酵母细胞的抗酮康唑，但那些没有进行转运改建为唑类抗18-20的原因，为CTY10-5D酵母新菌株是通过首先删除ERG3（ERG3Δ）获得，然后引入额外的缺失ERG11（ERG3Δ/ ERG11Δ）的基因通过同源重组。建筑细节在以前出版的手稿6被描述。 </li><li>第二天（早上），接种酵母（1:20）在5毫升YAPD和孵化通过不断搅拌（220转，30℃）来获取一个外径600〜0.2。 </li><li>沉淀细胞在微量离心2分钟2000转，并丢弃上清液。然后洗PELLE吨两次用无菌水，一次用0.1M的醋酸锂。 </li><li>最后一次洗涤后，弃去上清液，加入以下试剂，细胞沉淀:240微升PEG 3500 50％重量/体积，36微升1M的醋酸锂，50微升2毫克/毫升的水煮鲑鱼精子单链DNA，H 2 O的34微升。旋涡混匀，加1微克PSH-PXR和1微克pGADNOT-SRC-1的DNA混匀。 </li><li>传输组合，聚苯乙烯圆底管，搅动改造，混合（220转，30℃）30分钟。移动管子到42℃水浴中孵育另外的30分钟。 </li><li>离心管在2,000 rpm离心2分钟，除去上清液。用无菌水冲洗一次颗粒。 </li><li>吸取100微升无菌水进入管，通过手指轻轻重悬细胞攻和移液，然后镀上-His/-Leu降中（配方每升-His/-Leu差中的板材:20 G葡萄糖， 1.7克YNB，0.67克CSM-His/-Leu，5克硫酸铵，1.5％琼脂）和增量ubate在30℃下3-4天以分离转化体。 </li></ol> 3。 X-gal的筛选分析<ol><li>切硝酸纤维素膜的陪替氏培养皿的部分的大小。标签的硝酸纤维素膜上标记的方向。 </li><li>放置在酵母菌落预标记的硝酸纤维素膜，确保该膜是在与板介质的表面相接触。 </li><li>取下滤膜，将其放置菌落面朝上3毫米的滤纸，然后将膜和滤纸在-80℃下15分钟。 </li><li>让X-gal的解决方案。加50微升20毫克/毫升X-gal和15μl的β-巯基乙醇至5ml的Z-缓冲液（1升缓冲液含有16.1克的Na 2 HPO 4 7H 2 O，5.5克的NaH 2 PO 4·H 2 O，0.75克氯化钾，0.25克硫酸镁4•7H 2 O）。如果Z缓冲区保持在4℃，加入这些试剂之前，把它带到室温。 </li><li>将3毫米的滤纸两个圆在培养皿中，并用5毫升X-gal的溶液浸泡其中。排出多余的缓冲液从Petri培养皿并将其放置在硝酸纤维素膜上的菌落的一面朝上。 </li><li>盖上盖子，确保膜正与滤纸完全接触，这是完全湿透。包裹培养皿箔和孵化在37℃，30分钟至过夜。 </li></ol> 4。液体的β-半乳糖苷酶含量<ol><li>酵母生长在-His/-Leu差液体培养基中OD 600 0.7。 </li><li>转移1ml培养基至1.5 ml微量离心管中并离心以3,000 rpm持续2分钟。弃上清。 </li><li>加入1 ml的Z缓冲器中，以重悬沉淀，离心分离机在3000rpm下再持续2分钟。弃上清。 </li><li>加入150μlZ缓存与2ME重悬细胞沉淀，混匀再加入50微升氯仿和20微升0.1％SDS。大力旋涡振荡样品15秒。 </li><li>加入700μlONPG溶液（1毫克/毫升Z中的bufFER）。设置定时器，孵育在30℃下足够长的时间以使黄色开发并注意总的反应时间。 </li><li>加入500μl的1M的Na 2 CO 3使反应停止，并确定在420nm处的吸光度。空白是700微升ONPG溶液加500微升1米的Na 2 CO 3。 </li></ol>计算米勒单位如下:米勒单位=（A420 * 1,000）/（A600 *分*毫升） A420:吸光度单位在420纳米; A600:吸光度单位，在600纳米; 分钟 :反应时间（分钟）; 毫升 :在毫升反应体积

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没有利益冲突的声明。

在我们修改的酵母双杂交实验中，我们已经确定了对PXR 6酮康唑相互作用重要的残留物。因为SRC-1是共活化剂（和克隆到pGADNot矢量），我们还测试了SRC-1是否能当克隆到PSH载体系统激活lacZ的表达，以及是否会改变活动模式和/或影响的泄漏酵母双杂交实验。利用我们在ERG3Δ/ ERG11Δ酵母进行双杂交试验我们重新设计的质粒。和以前一样，我们表明lacZ基因表达（蓝色菌落）也诱导ERG3Δ...

在酵母双杂交（Y2H）法被广泛地用于发现蛋白质-蛋白质相互作用以及最近用于发现新颖的小分子干扰蛋白质-蛋白质相互作用复合体7，8，9，10，11。然而，这种分析的传统方法中，用于药物发现的或"命中"，不允许用于检测的化学品化合物的变构相互作用的残基中的蛋白-蛋白的表面，即改变仍然交互，并允许改变的残基11的询问时。事实上，这样的方法（次），如果制定可行，将使为别构相互作用的残基的蛋白质 - 蛋白质相互作用破坏关键的高通量评价一个听话的酵母系统。在药物发现的背景下，最直接的方式来建立化合物的相互作用与蛋白质会涉及结构测定（蛋白-抑制剂复合物如晶化）。这些方法暨bersome，用精心制作的资源和执行的每一个蛋白质结构研究它在技术上并不可行。 听话的基因药物筛选系统已经建立了细菌1，2和类似哺乳动物双杂交等模型系统。然而，这些系统需要最优化和类似Y2H替代系统仍然在药物发现的最测试。有迹象表明，包括灵敏度，并使用单数的方法13的相互作用的可靠性差的限制，然而，一个单一的Y2H测定法可以被修改来回答关于相互作用的残基的具体问题。在核受体的研究领域中，Y2H已被用来定义相互作用蛋白14，然而，这些蛋白质的相互作用很少被用于定义在其中的配体/受体拮抗剂与核受体-蛋白质复合物相互作用的性质。因此，我们的实验室集中精力定义一个方法，尤其是对于受体蛋白质，是不随时服从蛋白质组学为基础的方法，这将发掘新的配体/受体拮抗剂使用基于反向Y2H发现平台交互的残留物。 基于我们以前发现，酮康唑破坏PXR及其活化剂SRC-1，我们开发了一种新的反向酵母双杂交系统，使我们能够对PXR 6定义和查询酮康唑相互作用的残留物。我们的方法是基于酵母的GAL4的蛋白质，它由负责DNA结合和转录激活结构域可分离的属性。的PXR LBD蛋白被表达为融合到LexA的DNA结合结构域（DNA-BD），而全长共活化剂的SRC-1（类固醇受体共激活因子1）的蛋白质表达为融合至GAL4激活结构域（AD ）。 PXR和SRC-1融合蛋白之间的相互作用导致的含有报告基因的β-Lac的Ž已整合到酵母染色体组的GAL4结合位点的转录活化Ë。酮康唑，一个PXR受体拮抗剂，扰乱PXR和SRC-1相互作用15，16，17，我们可以检测PXR的相互作用和SRC-1的染色上筛选菌落用于X-gal的活动之后的存在或不存在酮康唑。 Y2H的原理图示于图1和实验过程总结于图2。

<table><tbody><tr><td>Yeast Strain CTY10-5d &lt;em&gt;erg3&lt;/em&gt;&amp;Delta;/&lt;em&gt;erg11&lt;/em&gt;&amp;Delta;</td><td>Our lab</td><td></td><td>CTY10-5d yeast was double knocked out &lt;em&gt;ERG3 &lt;/em&gt;and &lt;em&gt;ERG11&lt;/em&gt; (&lt;em&gt;erg&lt;/em&gt;&lt;em&gt;3&lt;/em&gt;&amp;Delta;/erg11&amp;Delta;) genes&lt;sup&gt;6&lt;/sup&gt; .</td></tr><tr><td>YPD Growth Medium</td><td>BD Biosciences</td><td>630409</td><td></td></tr><tr><td>Difco Yeast Nitrogen Base (YNB) w/o Amino Acids and Ammonium Sulfate</td><td>BD Biosciences</td><td>233520</td><td></td></tr><tr><td>Bacto Agar</td><td>BD Biosciences</td><td>214010</td><td></td></tr><tr><td>CSM-His/-Leu Complete Supplement Mixture</td><td>MP Biomedicals</td><td>4250-412</td><td></td></tr><tr><td>ONPG (o-Nitrophenyl &amp;Beta;-D- Galactopyranoside).</td><td>Sigma-Aldrich</td><td>N1127</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma-Aldrich</td><td>M6250</td><td></td></tr><tr><td>Luria Broth (LB)</td><td>Sigma-Aldrich</td><td>L3022</td><td></td></tr><tr><td>X-Gal</td><td>Fisher</td><td>BP-1615</td><td></td></tr><tr><td>Sonicated Salmon Sperm DNA boiled (10 mg/ml)</td><td>Life Technology</td><td>156-017</td><td></td></tr><tr><td>Ampicillin</td><td>Acros Organics</td><td>61177</td><td></td></tr><tr><td>Ketoconazole</td><td>Sigma-Aldrich</td><td>K1003</td><td></td></tr><tr><td>N,N-Dimethylformamide</td><td>Acros Organics</td><td>326871000</td><td></td></tr><tr><td>Lithium Acetate</td><td>Sigma-Aldrich</td><td>L4158</td><td></td></tr><tr><td>50% PEG-3350 solution, filter-sterilized</td><td>Sigma-Aldrich</td><td>P-3640</td><td></td></tr><tr><td>Nitrocellulose Membrane</td><td>Whatman</td><td>10402091</td><td></td></tr><tr><td>10 cm Petri Dish</td><td>Fisher</td><td>875712</td><td></td></tr><tr><td>5&#039;-ACCGGATCCCGATGAAGA AGGAGATGATCATGTCC-3&#039;</td><td>our lab</td><td></td><td>PXR LBD forward primer for pSH2-1</td></tr><tr><td>5&#039;-AGAGTCGACTCAGCTA CCTGTGATGCC -3&#039;</td><td>our lab</td><td></td><td>PXR LBD reverse primer for pSH2-1</td></tr><tr><td>5&#039;-TATAGC GGCCGCATGAGTG GCCTCGGGGACAGTTCATCC -3&#039;</td><td>our lab</td><td></td><td>SRC-1 forward primer for pGADNOT</td></tr><tr><td>5&#039;-GCGGTCGACTTATTCAGTCA GTAGCTG -3&#039;</td><td>our lab</td><td></td><td>SRC-1 reverse primer for pGADNOT</td></tr><tr><td>Platinum PCR Supermix</td><td>Invitrogen</td><td>11306-016</td><td></td></tr><tr><td>BamHI</td><td>our lab</td><td>R0136</td><td></td></tr><tr><td>SalI</td><td>our lab</td><td>R0138</td><td></td></tr><tr><td>NotI</td><td>our lab</td><td>R0189</td><td></td></tr></tbody></table>

reverse yeast two-hybrid system to identify mammalian nuclear receptor residues that interact with ligands and/or antagonists

由于药物代谢和炎症反应的重要调节器，孕烷X受体（PXR），它在疾病的病理生理过程中起重要作用联系起来代谢和炎症（ 如脂肪肝）1,2。出现了在激动剂配体为PXR的识别很大的进展，但是，也有类药拮抗剂和PXR 3,4,5其结合位点的有限的描述。一个关键的障碍已经无法有效地净化全长蛋白与拮抗剂的结构研究尽管PXR克隆和特征1998。我们实验室开发了一种新的高通量酵母基于双杂交法来定义的拮抗剂，酮康唑的，上PXR 6结合的残基。我们的方法包括创建突变库，将营救的单一突变对PXR的AF-2面有望与酮康唑交互的效果。事态抢救或"功能获得的"塞科第二突变可制成使得关于酮康唑对PXR的遗传相互作用和表面残基的结论是可行的。因此，我们开发PXR突变体及其共激活因子，SRC-1相互作用的高通量双杂交酵母屏幕。使用这种方法，其中的酵母进行了修改，以适应抗真菌药，酮康唑的研究中，我们能够证明对PXR的特定突变富集的克隆不能结合到酮康唑。通过反向逻辑，我们的结论是原来的残留物是直接的互动残酮康唑。此法是一种新型的，听话的基因检测，以屏幕上的核受体拮抗剂的表面结合位点。该测定法可以适用于任何药物，无论其细胞毒性潜力的酵母以及细胞蛋白（s）表示，不能使用标准结构生物学或基于蛋白质组学方法，研究。潜在的缺陷包括解释数据的（互补的方法很有用），可靠ANCE单Y2H的方法，在处理酵母或进行酵母双杂交试验的专业知识，并检测优化。

我们进行了一个实验，看看我们是否可以检测PXR和类固醇受体共激活因子-1（SRC-1）的协会的比色读数。由于酵母具有显著甾醇的生产，它先前已经表明，在酵母中的lacZ的表达，而不需要额外的外源性配体所诱导。我们发现，lacZ的表达（蓝色菌落）也感应出的酵母菌株转化的PXR和SRC-1，但是，没有诱导LacZ的表达（白色菌落）在酵母转化的空载体，PXR的或单独的SRC-1 （ 图3）。以确定酮...

Watch this Scientific Journal Video about Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists at JoVE.com

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

酮康唑结合并拮抗孕烷X受体（PXR）激活。 PXR突变体的酵母高通量筛选定义一个独特的区域为酮康唑约束力。这种酵母为基础的遗传学方法发现与具有表面结合位点相关联的配体小说核受体相互作用。

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 ...

reverse-yeast-two-hybrid-system-to-identify-mammalian-nuclear

Research

JoVE Journal

Biology

12.7K 次观看.  Albert Einstein College of Medicine. 酮康唑结合并拮抗孕烷X受体（PXR）激活。 PXR突变体的酵母高通量筛选定义一个独特的区域为酮康唑约束力。这种酵母为基础的遗传学方法发现与具有表面结合位点相关联的配体小说核受体相互作用。 

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

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

通过免疫共沉淀在核质馏分蛋白质相互作用的可视化和<em&gt;原位</em&gt;接近结扎分析

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

科研

生物化学

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between proteins such as transcription factors (TFs) and their DNA target. The method presented here utilizes the underlying concept of the Y1H but is modified further to study and validate protein complexes binding to their target DNA. Hence, it is referred to as the modified yeast one-hybrid (Y1.5H) assay. This assay is cost effective and can be easily performed in a regular laboratory setting. Albeit using a heterologous system, the described method could be a valuable tool to test and validate the heteromeric protein complex binding to their DNA target(s) for functional genomics in any system of study, especially plant genomics.

The modified yeast one-hybrid assay described here is an extension of the classical yeast one-hybrid (Y1H) assay to study and validate the heteromeric protein complex-DNA interaction in a heterologous system for any functional genomics study.

用于异构蛋白复合物 -  DNA相互作用研究的修饰的酵母一杂交系统

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between ...

Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay

Estrogen receptor alpha (ER&#945;) is an estrogenic ligand-dependent transcription regulator. The sequence of ER&#945; protein is highly conserved among species. It has been thought that the function of human and mouse ER&#945;s is identical. We demonstrate the differential 4-hydroxy-tamoxifen (4OHT) effect on mouse and human ER&#945; ligand-binding domain (LBD) dimerization activity using the mammalian two-hybrid (M2H) assay. The M2H assay can demonstrate the efficiency of LBD homodimerization activity in mammalian cells, utilizing the transfection of two protein expression plasmids (GAL4 DNA-binding domain [DBD] fusion LBD and VP16 transactivation domain [VP16AD] fusion LBD) and a GAL4-responsive element (GAL4RE) fused luciferase reporter plasmid. When the GAL4DBD fusion LBD and the VP16AD fusion LBD make a dimer in the cells, this protein complex binds to the GAL4RE and, then, activates a luciferase gene expression through the VP16AD dependent transcription activity. The 4OHT-mediated luciferase activation is higher in the HepG2 cells that were transfected with the mouse ER&#945; LBD fusion protein expression plasmids than in the human ER&#945; LBD fusion protein expression plasmid transfected cells. This result suggests that the efficacy of the 4OHT-dependent mouse ER&#945; LBD homodimerization activity is higher than human ER&#945; LBD. In general, the utilization of the M2H assay is not ideal for the evaluation of nuclear receptor LBD dimerization activity, because agonistic ligands enhance the basal level of the LBD activity and that impedes the detection of LBD dimerization activity. We found that 4OHT does not enhance ER&#945; LBD basal activity. That is a key factor for being able to determine and detect the 4OHT-dependent LBD dimerization activity for successfully using the M2H assay. ER&#945; LBD-based M2H assays may be applied to study the partial agonist activity of selective estrogen receptor modulators (e.g., 4OHT) in various mammalian cell types.

We present a method for analyzing the 4-hydroxy-tamoxifen-dependent estrogen receptor alpha ligand-binding domain dimerization activity using the mammalian two-hybrid assay.

利用哺乳动物双杂交法检测雌激素受体α的配联域二维化活性

Estrogen receptor alpha (ER&#945;) is an estrogenic ligand-dependent transcription regulator. The sequence of ER&#945; protein is highly conserved among ...

癌症研究

Yeast As a Chassis for Developing Functional Assays to Study Human P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

酵母作为开发功能分析研究人类P53的机箱

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

A High-Throughput Screening Approach for Evaluating the Binding Affinity of Environmental Pollutants to Nuclear Receptors

Increasing levels of compounds have been detected in the environment, causing widespread pollution and posing risks to human health. However, despite their high environmental occurrence, there is very limited information regarding their toxicological effects. It is urgent to develop high-throughput screening (HTS) methods to guide toxicological studies. In this study, a receptor-ligand binding assay using an HTS system was developed to determine the binding potency of environmental pollutants on nuclear receptors. The test is conducted using a microplate reader (i.e., a 96-well plate containing various chemicals) by measuring the fluorescence polarization (FP) of a specific fluorescent probe. This assay consists of four parts: the construction and transformation of recombinant vectors, the expression and purification of the receptor protein (ligand-binding domain), receptor-probe binding, and competitive binding of chemicals with the receptor. The binding potency of two environmental pollutants, perfluorooctanesulfonic acid (PFOS) and triphenyl phosphate (TPHP), with peroxisome proliferator-activated receptor gamma (PPAR&#947;) was determined to illustrate the assay procedure. Finally, the advantages and disadvantages of this method and its potential applications were also discussed.

This protocol describes a high-throughput screening system that uses fluorescence polarization of a specific fluorescent probe binding to a nuclear receptor as a readout for screening environmental pollutants.

Increasing levels of compounds have been detected in the environment, causing widespread pollution and posing risks to human health. However, despite ...

环境科学

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 ...

生物学

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 ...

A BW Reporter System for Studying Receptor-Ligand Interactions

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native receptor and the ligand are challenging since the ligand of a specific receptor may be unknown and experimental procedures with the native ligand can be technically complicated. To address these obstacles, we describe a reporter system to detect the binding and activation of a specific receptor by a ligand of interest. In this reporter system, the extracellular domain of a specific receptor is conjugated to mouse CD3&#950; and this chimeric protein is then expressed in mouse BW cells. These transfected BW cells can then be incubated with different targets (e.g., cells or antibodies). Activation of a transfected receptor leads to the secretion of mouse interleukin-2 (mIL-2) which can be detected by enzyme-linked immunosorbent assay (ELISA). This reporter system has the advantages of being sensitive and specific to a single receptor. In addition, the activation level of a specific receptor can easily be quantified and can be used even in cases where the ligand of the receptor is unknown. This system has been implemented successfully in many of our studies to characterize receptor-ligand interactions. We have recently employed this system to study the activation of human Fc&#947; receptors (Fc&#947;Rs) by different monoclonal anti-CD20 antibodies in clinical use.

This protocol describes how to establish a reporter system that can be used to identify and quantify receptor-ligand interactions.

一种研究受体配体相互作用的 bw 报告系统

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native ...

使用酵母双杂交技术的生物分子相互作用研究 

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

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the role(s) and function(s) of a specific protein-protein interaction is to compare the phenotype of the wild-type (with the relevant protein-protein interaction) and those of mutants that lack the relevant interaction. Therefore, if such mutants can be isolated, they will help to elucidate the related biological processes. The yeast two-hybrid (Y2H) procedure is a powerful approach not only to detect protein-protein interactions but also to isolate interaction-null/impaired mutants. In this article, a protocol is presented to isolate interaction-null/impaired mutants using Y2H technology. First, a mutation library is constructed by combining the polymerase chain reaction and efficient seamless cloning technology, which efficiently excludes the empty vector from the library. Second, interaction-null/impaired mutants are screened by the Y2H assay. Because of a trick in the Y2H vector, undesired mutants, such as those with frameshift and nonsense mutations, are efficiently eliminated from the screening process. This strategy is simple and can, therefore, be applied to any combination of proteins whose interaction can be detected by the two-hybrid system.

作者聚焦:增强酵母中相互作用零突变体的分离

The interactions of biomolecules, such as protein-protein interactions, are the molecular basis of biological functions. If interaction-null/impaired mutants that specifically lack the relevant interaction can be isolated, they will greatly help to understand the function(s) of this interaction. This article presents an efficient way to isolate interaction-null/impaired mutants.

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 ...

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

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