这项工作是由NIH的研究补助金K01AR053210，R01AR061484和国家牛皮癣基金会的赠款。

1。背景资料酵母双杂交系统是一个功能强大的基因技术，用于发现蛋白质相互作用16， 17。 2混合动力系统，如lexA为基础的系统，SOS招聘系统，细菌或哺乳动物细胞为基础的2混合动力车，几种商业可用，本文特别侧重于最常用的GAL4基于修改酵母双杂交系统。简而言之，该方法是基于负责可分离DNA结合和转录激活域组成的酵母GAL4蛋白的属性。诱饵蛋白表达作为一个融合的GAL4 DNA结合结构域（DNA - BD的），而猎物蛋白GAL4激活结构域（AD）的融合表达。诱饵和猎物的融合蛋白之间的相互作用，导致转录激活GAL4 -约束力的网站含有报告基因整合到酵母克enome。 图所示的Y2H原则。图1和实验过程中总结。 2。 2。所需的材料和解决方案<ol><li> YPD生长培养基（融合蛋白胨，酵母提取物，和最佳比例增长最酿酒酵母株葡萄糖）。 </li><li>最小的SD基地（最小合成（SD）的基地包括一个酵母氮基，硫酸铵，和碳源，葡萄糖差（DO）的补充，可以添加到最小的SD基地，使一种人工合成的，缺乏指定的定义介质营养素）。 </li><li>亮氨酸/色氨酸差（DO）的补充（含所有必需氨基酸除亮氨酸和色氨酸） </li><li> -His/-Leu/-Trp/-Ura差（DO）的补充文件（含所有必需氨基酸除亮氨酸，色氨酸，组氨酸，和尿嘧啶） </li><li>卢里亚肉汤（LB）（蛋白胨10克/ L，酵母提取物5 g / L的氯化钠5克/升） </li><li> X-半乳糖（5 - 溴-4 - 氯-3 - 吲哚-β- D -半乳糖苷）解决方案：20毫克/毫升原液在N，N -二甲基甲酰胺编写</li><li> 10X的TE（100毫米的Tris - HCl（pH值7.5），10 mM的EDTA，蒸压） </li><li>超声鲱鱼或鲑鱼精DNA，煮沸（10毫克/毫升） </li><li> 10X LiAc（1个M醋酸锂，蒸压） </li><li> 50％PEG - 3350溶液，过滤消毒</li><li> 3 - 氨基- 1，2，4 - 三唑（3AT） </li><li>卡那霉素</li><li>氨苄青霉素</li><li> ELECTROMAXDH5α细胞</li><li> Z缓冲区：16.1克的Na2HPO4 · 7H2O（或8.52 g无水），5.5克磷酸二氢钠水（或4.8 ganhydrous），0.75克氯化钾，0.246克硫酸镁7H2O（或0.12 g无水），在1升高压灭菌蒸馏水溶解和调整，以pH值7.0。 </li></ol> 3。诱饵代（pDBLeu - PGRN） 萨尔我不是我的pDBLeu载体的网站（在ProQuest双杂交系统，Invitrogen公司）定向克隆到一个cDNA片段的编码PGRN缺乏信号肽（aa21 - 588），保持相同的翻译为GAL4 DNA结合结构域的阅读框产生pDBLeu - PGRN。 <ol><li>健全的PGRN cDNA片段描述PCR以上使用寡核苷酸引物设计包含的酶切位点（萨尔我在5&#39;端和我的3&#39;end），以便在帧融合。 </li><li>凝胶纯化PCR产物，消化限制性内切酶萨尔我，不是我。 准备具有相同的限制性内切酶双酶切的DB载体pDBLeu。 </li><li>结扎成线性pDBLeu载体的限制PGRN片段，转化到DH5α中选择为LB +卡那霉素25微克/毫升。 </li><li>验证DNA测序构建的校正。 </li></ol> 4。诱饵质粒的小规模改造<ol><li>接种Mav203（Invitrogen公司）的殖民地的YPD 5毫升，摇匀一夜之间在30 ° C。 </li><li>稀释在50毫升的YPD培养过夜。长出一个添加itional 2-4小时。 </li><li>颗粒的细胞在室温下5分钟3000转。蒸压，蒸馏水40毫升的重悬沉淀。 </li><li>重新颗粒细胞。悬浮在我，在室温10分钟孵育2溶液。 解决方案：0.5毫升10xLiAc 10xTE，0.5毫升，4毫升H 2 O </li><li>分配管的DNA：2-3μLpDBLeu - PGRN（0.25μg/μl）和10μL变性，剪切鲑鱼精子DNA（10μg/μl）。加入100μL的酵母细胞，拌匀。 </li><li>加入700μl的解决方案二，拌匀。在30℃孵育30分钟。解决方案二：0.2毫升10xLiAc，10xTE 0.2毫升，1.6毫升50％PEG3350。 </li><li>热休克在42 ° C为15分钟。 </li><li>颗粒为2分钟。弃上清。在200μL灭菌蒸馏水重悬沉淀。 </li><li> SD - Leu平板上连续稀释板暂停。在30 ° C孵育板为2-3天。 </li></ol> 5。诱饵验证在执行YEA之前ST双杂交筛选，测试pDBLeu PGRN自激活，并确定HIS3报告基因的基础表达水平。该测试确定是否诱饵激活转录自激活是否可以抑制剂瓦解。 3 -氨基- 1，2，4 -三唑（3 - AT的）是一种HIS3基因产品的竞争性抑制剂，可用于滴定 HIS3表达组氨酸缺陷型媒体的增长所需的最低水平。 <ol><li> pDBLeu - PGRN转换成酵母Mav203应变，板到SD - Leu平板的转型，并孵育48-72 h 30 ° C。 </li><li>使用高压灭菌牙签上，在每个转换补丁的殖民地SD - LEU板含有3 - AT浓度为10毫米，25毫米，50毫米，75毫米，100毫米。 3 - AT的是HIS3酶和展现自我激活的唯一诱饵的竞争性抑制剂，将能够成长中存在的3 - AT。 </li><li>诱饵菌株生长板containi吴100毫米3 - AT是不适合使用双杂交筛选。对于可使用毒饵，使用的3 - AT的浓度，抑制细胞生长的最低。在许多情况下，3 - AT的25毫米用于筛查。 </li></ol> 6。 cDNA文库筛选如上所述，使用一个小规模的的改造，pDBLeu - PGRN质粒引入MaV203。要引入MaV203（pDBLeu PGRN）pPC86库（Invitrogen公司），下面描述的步骤通常 ​​为〜4 × 10 4菌落质粒文库DNA与0.5微克。因此，将需要2.5 × 10 6酵母转化〜30.0微克pPC86 cDNA文库质粒DNA，25转换，以及50个10厘米的板（SD - LEU - TRP -浦3 AT ）。 <ol><li>板（50以下步骤），准备在适当数量的10厘米SD - LEU -色氨酸，他的浦3。还准备估算转化的数量至少有四个10厘米SD -亮氨酸色氨酸板块。 </li><li>变换成的图书馆MaV203含有pDBLeu - PGRN按程序介绍如下。 <ol><li>暂停MaV203几个孤立的殖民地〜100μL灭菌蒸馏水（pDBLeu PGRN），并传播到10厘米的SD - LEU板。 SD - Leu的第二板重复的过程。孵育18-24 h两个板块在30 ° C。 </li><li>刮掉，并完全停止在10毫升灭菌蒸馏水细胞。加入足量的细胞悬液500毫升的烧瓶中的液体YPD培养基给外径600〜0.1。 </li><li>验证外径为0.1〜接种后。 </li><li> 30摇外径600℃直至达到〜0.4。 </li><li>准备好新鲜： <ol type="a"><li> 110毫升1X TE / LiAc相结​​合，11毫升的TE 10X 10X LiAc 11毫升，88毫升灭菌水。 </li><li> 16毫升PEG / LiAc 1.6毫升的TE 10X，10X LiAc 1.6毫升，12.8毫升50％的PEG - 3350相结合。 </li></ol></li><li>在室温下5分钟沉淀细胞在3000克。 </li><li>丢弃吨他上清，轻轻重悬沉淀吹打起来，在100毫升灭菌蒸馏水，在室温下。 </li><li>在3000克离心机在室温下5分钟。 </li><li>弃掉离心细胞上清，重悬细胞沉淀于50 ml 1X TE / LiAc解决方案。 </li><li>在3000克离心机在室温下5分钟。 </li><li>取出上清和沉淀重悬在2.5毫升的1X TE / LiAc解决方案的最终体积。 </li><li>执行25转换。将2.5毫升的细胞，125μL（10μg/μl）变性，剪切鲑鱼精子DNA，cDNA文库和30微克。轻轻地上下吹打混合。添加15毫升的PEG / LiAc解决方案，并轻轻混匀。分装成25各700μL灭菌的1.5 ml离心管。 </li><li>孵育在30℃水浴30分钟。 </li><li>热休克在42℃水浴15分钟。 </li><li>在6000克离心机在室温下1分钟。小心取出超游泳。轻轻地上下吹打重悬在400μL灭菌蒸馏水每个颗粒。 </li></ol></li><li>板200μL改造从改造到10厘米的单SD -亮氨酸色氨酸他浦3 AT（3AT浓度：25毫米）的每个混合板使用的传播栏，使25蒸压1.5毫升的离心管，可以使50板（SD - Leu的色氨酸他浦3 AT）。 </li><li> 5-10天在30 ° C孵育板</li><li>为了估计反应的转化效率，一个反应（1:40; 1:400和1:4000）的串行稀释SD -亮氨酸色氨酸板镀。经过连续3天在30℃，计数菌落数和总人数的计算转化的孵化。 </li></ol> 7。 X - gal的检测<ol><li>传输殖民地YPD板;孵育30 ° C过夜。 </li><li>广场圆润的YPD板上硝酸纤维素膜，确保有膜和YPD板之间无气泡。 1-2分钟后，马柯确保所有殖民地被转移到膜（硝化棉纸）。 </li><li>船在用铝箔纸将膜殖民地的一面向上。 </li><li>膜放入液氮，液氮等待一两分钟20秒，并淹没。 </li><li>取出膜解冻。 </li><li>同时混合下列试剂： 1.5毫升ž缓冲区 X - gal的20μL（20毫克/毫升） </li><li>掉落Z缓冲区/ X - gal的培养皿中，最重要的是地方的Whatman纸。 </li><li>小心地放置在顶部的Whatman纸硝化纤维纸。 </li><li>在37℃孵育，直到出现蓝色菌落。 </li></ol> 8。 Retransformation检测从库中筛选分离的猎物融合蛋白（AD - Y），应保留诱饵融合蛋白（DB - X）的相互作用，以诱导的报告基因，和猎物的克隆和诱饵retransformation建设成酵母可以进一步消除误报和方便添加itional分析。 <ol><li>隔离酵母菌株的质粒DNA可能含有蛋白质相互作用。 </li><li>转换成大肠杆菌DNADH5α细胞，在LB板的转换100μg/ mL氨苄青霉素板选择性分离pPC86 cDNA文库，孵育过夜，于37 ° C。 </li><li>从板块中挑选几个菌落于LB 100μg/ mL氨苄青霉素肉汤。 </li><li>准备小量DNA和检查由用Sal I和Not一双酶切分析</li><li>合作改造的猎物质粒和诱饵质粒进入MaV203，板改造的混合物，SC -亮氨酸色氨酸板，并为2-3天，在30 ° C孵育</li><li>选择SC -亮氨酸色氨酸板从三个不同的殖民地，进行X - gal的含量。 </li></ol> 9。测序和生物信息学分析序列可能真阳性克隆中分离的质粒DNA，比较这些序列在GenBank中使用的BLAST程序，并确定这两个杂交对应于已知基因的克隆。测序数据显示，获得上述12阳性细胞表面TNFR2（TNFRSF1B/CD120b加入＃NM_130426）。此外，PGRN和肿瘤坏死因子受体之间的相互作用进行了验证，使用各种蛋白质相互作用的分析，在体外包括固相结合法，免疫共沉淀，表面等离子体共振分析， 流式细胞仪检测14。 10。代表性的成果甄别的流程图如图概述 。 3。通常50-100阳性克隆的候选人将获得在这一步。我们初步分离出250万PGRN诱饵筛选转化之间的54个阳性克隆候选人。阳性克隆的候选人，然后通过X - gal的检测验证。通常情况下，约50％的假阳性克隆，通过X - gal的检测中删除。我们获得23坦率积极的克隆<html>阿泰在这一步PGRN诱饵（ 图4）。猎物的克隆和诱饵构建成酵母Retransformation进一步消除误报和克隆，仍然激活基因可能代表真阳性。通常情况下，约50％的阳性克隆的候选人将被删除。我们终于分离出12个阳性克隆，酵母PGRN互动。 <img alt="图1" src="/files/ftp_upload/3562/3562fig1.jpg" /> 酵母双杂交系统的原理图1 。 <img alt="图2" src="/files/ftp_upload/3562/3562fig2.jpg" /> 图2管道确定蛋白用酵母双杂交系统的结合伙伴 。 <img alt="图3" src="/files/ftp_upload/3562/3562fig3.jpg" /> 图3。流量图表筛选酵母双杂交文库。rge.jpg“目标=”_blank“&gt;点击这里查看一个全尺寸的版本，这个形象。 <img alt="图4" src="/files/ftp_upload/3562/3562fig4.jpg" /> 图4。β-半乳糖苷酶检测阳性克隆的候选人。库屏幕获得一个阳性克隆转移到YPD板，并在30 ° C过夜培养; B，YPD板所有殖民地被转移到硝酸纤维素膜和β-半乳糖苷酶测定执行。

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progranulin+Resolves+Inflammation.">Progranulin Resolves Inflammation.</a> Science. 332, 427-428 (2011).</li><li>Hollenberg, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+new+family+of+tissue-specific+basic+helix-loop-helix+proteins+with+a+two-hybrid+system.">Identification of a new family of tissue-specific basic helix-loop-helix proteins with a two-hybrid system.</a> Mol. Cell. Biol. 15, 3813-3822 (1995).</li><li>Vojtek, A. B., Hollenberg, S. M., Cooper, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+Ras+interacts+directly+with+the+serine/threonine+kinase+Raf.">Mammalian Ras interacts directly with the serine/threonine kinase Raf.</a> Cell. 74, 205-214 (1993).</li></ol>

没有利益冲突的声明。

酵母双杂交筛选已被证明是一个有效的工具，在确定蛋白质相互作用16，17。与其他方法确定蛋白结合的合作伙伴，如生化合作，纯化和蛋白质芯片，酵母双杂交系统是一个敏感的遗传筛选非常高的数字编码序列在一个相对简单的的实验方法，可使用;此外，它在体内相互作用检测，也不需要复杂的蛋白质纯化。当然酵母双杂交系统有其局限性，例如，需要翻译后修饰，融合蛋白折叠的不足...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> ProQuest双杂交系统</td><td> Invitrogen公司</td><td> 10835 </td></tr><tr><td> YPD生长媒介</td><td> Clontech公司</td><td> 630409 </td></tr><tr><td> YPD琼脂媒介</td><td> Clontech公司</td><td> 630410 </td></tr><tr><td>最小的SD琼脂基地</td><td> Clontech公司</td><td> 630412 </td></tr><tr><td> - Leu的请补充</td><td> Clontech公司</td><td> 630414 </td></tr><tr><td> -Leu/-Trp DO补编</td><td> Clontech公司</td><td> 630417 </td></tr><tr><td> -His/-Leu/-Trp/-Ura DO补编</td><td> Clontech公司</td><td> 630425 </td></tr><tr><td> 3 - 氨基- 1，2,4 - 三唑（3AT） </td><td> Sigma - Aldrich公司</td><td> A8056 </td></tr><tr><td>卢里亚肉汤（LB） </td><td> Sigma - Aldrich公司</td><td> L3022 </td></tr><tr><td> X - Gal的</td><td> Invitrogen公司</td><td> 15520-034 </td></tr><tr><td>超声波处理鲑鱼精子DNA </td><td> Stratagene公司</td><td> 201190 </td></tr><tr><td>氨苄青霉素</td><td> AMRESCO </td><td> 0339 </td></tr><tr><td>硫酸卡那霉素</td><td> Invitrogen公司</td><td> 11815-024 </td></tr><tr><td>亚克隆效率™DH5α™感受态细胞</td><td> Invitrogen公司</td><td> 18265-017 </td></tr><tr><td> Trizma ®基地</td><td> Sigma - Aldrich公司</td><td> T6066 </td></tr><tr><td>醋酸锂</td><td> Sigma - Aldrich公司</td><td> L4158 </td></tr><tr><td>乙二胺四乙酸</td><td> Sigma - Aldrich公司</td><td>教育署</td></tr><tr><td>硝酸纤维素膜</td><td> Bio - Rad公司</td><td> 162-0115 </td></tr><tr><td> 10厘米的培养皿</td><td> ITI的科学</td><td> CT - 903 </td></tr><tr><td>培养箱（30℃） </td><td> ATR（Ecotron） </td><td></td></tr></table>

modified yeast-two-hybrid system to identify proteins interacting with the growth factor progranulin

Progranulin（PGRN），又称granulin epithelin前体（GEP），是一个593个氨基酸的自分泌生长因子。 PGRN是已知的各种生理和疾病过程中发挥了关键作用，包括早期胚胎发育，伤口愈合，炎症2，3，和主机防御4。 PGRN也可以作为一种神经营养因子，并在PGRN基因导致部分损失的PGRN蛋白引起的额颞叶痴呆6，7突变。我们最近的研究作为重要的调节软骨发育和退化8-11导致PGRN隔离。虽然PGRN，发现近二十年前，起着至关重要的作用，在多种生理和病理条件下，努力开拓PGRN的行动，并了解所涉及的机制已明显阻碍我们无法确定其结合受体（S）。为了解决这个问题，我们开发了莫迪fied酵母双杂交（MY2H）方法的基础上最常使用的GAL4基于2混合动力系统。 MY2H与传统的酵母双杂交屏幕相比，极大地缩短了屏幕的过程，并降低了假阳性克隆数。此外，这种方法是重复的，可靠的，我们已经成功地采用这个系统，在隔离各种诱饵的结合蛋白，包括离子通道，细胞外基质蛋白10，13，和生长因子14。在本文中，我们详细描述了作为一个例子，LED作为第一个已知的PGRN相关受体14日，15 TNFR2识别使用PGRN这一MY2H实验过程。

Watch this Scientific Journal Video about Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin at JoVE.com

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

我们已经修改了传统的酵母双杂交筛选，确定蛋白质相互作用的一种有效的遗传工具。此修改的过程明显缩短，减少了工作量，最重要的是，降​​低了误报数量。此外，这种方法是重复的，可靠的。

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 ...

modified-yeast-two-hybrid-system-to-identify-proteins-interacting

Research

JoVE Journal

Biology

28.6K 次观看.  NYU Hospital for Joint Diseases. 我们已经修改了传统的酵母双杂交筛选，确定蛋白质相互作用的一种有效的遗传工具。此修改的过程明显缩短，减少了工作量，最重要的是，降​​低了误报数量。此外，这种方法是重复的，可靠的。

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

Growth Factor-Coated Bead Placement on Dorsal Forebrain Explants

This video demonstrates two methods for preparing and placing beads, which have been coated with growth factor, on explants of the developing cerebral cortex. These beads can be used to induce spatially restricted gene expression on developing neural tissue such as forebrain explants. Methods are given for using both Affi-Gel beads and heparin acryllic beads.

涂层生长因子-珠放在背的前脑植

科研

生物学

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

配售早期鸡胚生长因子包被的磁珠

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

Preparing T Cell Growth Factor from Rat Splenocytes

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. Addition of interleukin 2 to the culture media during the expansion phase is necessary to prevent cell death and sufficient to maintain short-term T cell lines but has been shown to increase Th1 polarization 3. Replacement of interleukin 2 by T cell growth factor (TCGF) which contains a mix of cytokines is more effective than interleukin 2 in maintaining long-term T cell lines in vitro 3. Moreover, TCGF can easily be prepared in large amounts in the laboratory and is much cheaper than recombinant interleukin 2. 
Here, we show how to prepare TCGF from rat splenocyte culture supernatants. For this procedure, we harvest spleens from naive Lewis rats euthanized for thymus and blood collection. We prepare single-cell suspensions from the spleens, lyze the red blood cells by osmotic shock, and seed the splenocytes in culture medium. The cells are stimulated with concanavalin A, a mitogen that non-selectively activates all rat T lymphocytes, inducing the production of cytokines. The culture supernantant is collected 48 hours later andexcess concanavalin A is bound to alpha methyl mannoside to prevent it from activating T cell lines to which TCGF will be added. The TCGF is then sterile-filtered, aliquoted, and stored at -20&deg;C.

We describe the preparation of T cell growth factor used for the in vitro expansion of antigen-specific rat T lymphocyte lines.

准备从大鼠脾细胞T细胞生长因子

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. ...

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

蛋白质组学鉴定蛋白质，P2X2配体门控的阳离子通道交互

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

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 ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR剪辑 - 一个方法来确定全转录组学，RNA结合蛋白的结合位点

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi筛选识别胚后的表型 C。线虫</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

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 ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

捕获复合质谱 - 一个强大的工具来确定新型C-二GMP效应蛋白

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

非洲爪蟾作为一种模型来确定翻译减值

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...