Name: a modified yeast-one hybrid system for heteromeric protein complex-dna interaction studies
Uploaded: 2017-07-24T15:00:00.000+00:00
Duration: 10 min 47 s
Description: Watch this Scientific Journal Video about A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies at JoVE.com

我们感谢SS Wang，Amar和A. Galla对手稿的批判性阅读。本出版物报道的研究得到了美国国家卫生研究院综合医学科学研究所的支持，获得了授权号RO1GM067837和RO1GM056006（SAK）。内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方观点。

<html> 1.构建和记录质粒制备<ol><li> PCR扩增待分析的靶DNA的启动子区/"片段"（优选约250-500bp），以使用大肠杆菌 （TOP10）进一步克隆入入载体。 </li><li>一旦测序确认，亚克隆阳性克隆在7如前所述8，9，10，11兼容pGLacZi表达载体。 </li><li>通过pGLacZi的同源重组变换启动子融合（YM4271）的酵母菌株来产生酵母报告菌株如前面12,13说明。这里没有描述具有DNA区域的酵母菌株与培养基配方的转化和确认步骤，因为步骤类似于经典的Y1H方案屁股= "外部参照"&gt; 9，10，11，12，13。所述PEXP-AD502对照质粒可以同时quantifiying如前所述8β半乳糖苷酶活被用于归一化目的。 </li><li>克隆感兴趣的TF或蛋白质，随后将相互作用的蛋白质伴侣克隆到pDEST22和pDEST32ΔDBD（pDEST32减去DNA结合域）表达载体中。转化的酵母启动子片段和TF克隆随后在方案中进一步使用。 </li></ol> 2.修改Y1.5H协议<ol><li>在第1天（下午），从培养皿或甘油浆中的SD-Ura中的 5mL培养物开始，并在30℃振荡器中孵育过夜。 注意:这种培养可以从新鲜条纹板或直接从酵母的25％或30％甘油原料开始用DNA片段克隆。 </li><li>在第2天（下午），用500μL过夜培养物接种10mL YPDA培养基，并在30℃振荡器中孵育过夜。 </li><li> 第3天（清晨和整个下午）:准备感受态细胞（清晨）。 <ol><li>用2 mL过夜培养物接种100mL的YPDA培养基，并在30°C振荡器中孵育。生长这种新鲜的培养物，直到OD 600达到0.4-0.8（3-5小时）。 注意:检查过夜培养物的OD 600 ，并确保此步骤的起始点OD 600为0.1 - 0.2。使用过度培养或培养不足的培养物可能会延长实验。 </li><li>在1000xg和21℃下离心5分钟。丢弃上清液。 </li><li>使用旋涡或上下移液将颗粒重建在10mL无菌水中。 </li><li>在1000xg和21℃下离心5分钟。丢弃上清液。 </li><li> Reconstitu使用涡旋或吸管上下游，在2mL Tris-乙二胺四乙酸（TE）/乙酸锂（LiAc）中沉淀。 注意:请参见表1中 TE / LiAc的配方。 </li><li>离心5分钟1000 xg和21°C。丢弃上清液。 </li><li>在4 mL TE / LiAc + 400μL鲑鱼精DNA（10 mg / mL）中上下重复悬浮沉淀，并进行下一步。 </li></ol></li><li> 共同转化:热休克细胞（下午晚些时候） <ol><li>在96孔混合板（U底）中分配每孔20μL的细胞。 </li><li>向孔中加入每个pDEST22:TF质粒，pDEST32ΔDBD:TF质粒和pEXP-AD502加pDEST32ΔDBD:TF（归一化对照）150ng（〜1.5μL） 注意:为了成功共同转化，pDEST22:TF和pDEST32ΔDBD:TF质粒各约150ng，预期最佳结果。可以随构建体和质粒表达而变化需要每个实验进行优化。另外一个控件pDEST22-TF加pDEST32deltaDBD也可以由用户自行决定。 </li><li>每孔加入100μLTE / LiAc /聚乙二醇（PEG）（ 混合三次 ）。 注意:请参见表1中 TE / LiAc / PEG的配方。 </li><li>用透气密封盖住板，并在30℃下孵育20-30分钟（可以更长时间）（无需搅拌）。 </li><li>在42℃下热冲击20分钟（ 精确 ）。 </li></ol></li><li> 平板细胞 <ol><li>在1000xg和21℃下离心5分钟。使用多通道移液器去除上清液。加入110μLTE，混匀。 </li><li>在1000xg和21℃下离心5分钟。使用多通道移液管去除100μL上清液。用打孔机重新悬挂颗粒。 </li><li>将细胞转移到SD-Trp-Leu琼脂平板上。孵化板在30°C直到下一步。 </li></ol></li><li> 第5天（下午）:使用打孔机/微孔板复制器将细胞转移到新板上。 <ol><li>使用多通道移液管，用无菌的96孔混合板（U底）填充50μLTE。在TE中预冲洗打孔机。 注意:打孔机或微孔板复制器应在此步骤之前进行灭菌，随后在方案中进行灭菌。可以应用将浸入乙醇（100％，不分子生物学级），然后将其燃烧到火焰上的冲孔机的常见方法。等待1分钟冷却冲孔针。 注意:如果在长凳上进行灭菌;确保工作台用乙醇预先清洁，周围没有任何可燃物质。穿戴适当的个人防护装备（PPE）。 </li><li>打开殖民地并在TE填充的板块中重新挂起。 注意:如果平板生长平稳，可以直接打孔到TE填充板或替代地，应使用消毒牙签将单个菌落（在多个菌落的情况下）捡拾到TE填充的板上，随后打孔机可以用于下一步骤。 </li><li>转移到新的SD-Trp-Leu琼脂平板上，以获得最佳的表面覆盖率。孵育板在30°C直到下一步。 </li></ol></li><li> 第7天（下午）:开始培养以测定β-半乳糖苷酶活性。 <ol><li>使用多通道移液管，用无菌96孔混合板（U底）填充50μL的SD-Trp-Leu培养基。 </li><li>使用多通道移液管，用180μL的SD-Trp-Leu培养基填充无菌96-深孔块。 </li><li>将培养基中的冲孔机预先浸湿，并将菌落从板转移到50μL的培养基。 </li><li>将20μL酵母转移到180μL的SD-Trp-Leu培养基中，并用透气密封件密封块。孵化在30℃振荡器下36小时。 </li></ol></li><li> 第9天（早晨）:开始用于酶测的培养。 <ol><li>将100μL的培养物转移到无菌96深孔块中的500μLYPDA培养基中。 </li><li>用透气密封盖住灭菌的深孔板，并在30℃下以200rpm搅拌孵育3-5小时（直至OD 600 0.3-0.6）。 </li><li>使用多通道移液管在分光光度计平板上重新悬浮培养并转移125μL（测量OD 600 ）。 注意:注意:如果OD 600低于0.3 - 0.6，请勿分配最后一滴以避免气泡，请根据读数将剩余的细胞孵育1 - 2小时。在此步骤中使用的125μL培养基可以丢弃或转移回原始的深井区块，用户可以自行决定。 </li><li>在3000×g和21℃下离心深井块中剩余的细胞10分钟。去除上清液t反转。 </li><li>加入200μLZ缓冲液并涡旋。 注意:请参见表1中 Z缓冲区的配方。 </li><li>在3000 xg和21℃下离心5分钟。倒置去除上清液。 </li><li>加入20μLZ缓冲液和涡旋。 </li><li>用可以抵抗冻融循环的密封箔覆盖板。在通风柜中使用液氮进行4个循环的冷冻/融化，然后在水浴中进行42℃（每次2分钟）。 </li><li>加入200μLZ-缓冲液/β-巯基乙醇（β-ME）/邻硝基苯基-β-半乳糖苷（ONPG）（12mL，21μL，8.4mg分别得到20mL溶液; 总是制备新鲜的 ）。 注意:ONPG需要一些时间才能正确溶解，以便在达到此步骤前一小时准备溶液。 ONPG作为测定β-半乳糖苷酶活性的底物。 </li><li>在30°C下孵育高达17 - 24小时（如果颜色发展较早，请检查继续下一步）。 </li></ol></li><li> 第10天（早晨）:停止反应并测量。 <ol><li>加入110μL1M Na 2 CO 3以停止反应并记录时间。 </li><li>涡旋并在3000 xg和21℃下离心10分钟。 </li><li>使用多通道取125μL上清液并测量OD 420 。 注意:注意，不要分配最后一滴以避免气泡。 </li><li>在电子表格中计算β-gal活性:1000×OD 420 /（时间（分钟）×体积（mL）×OD 600 。 </li></ol></li></ol>

<ol>
	<li>Saghbini, M., Hoekstra, D., Gautsch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Media+formulations+for+various+two-hybrid+systems.">Media formulations for various two-hybrid systems.</a> Methods Mol Biol. 177, 15-39 (2001).</li><li>Reece-Hoyes, J. S., Marian Walhout, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+one-hybrid+assays:+A+historical+and+technical+perspective.">Yeast one-hybrid assays: A historical and technical perspective.</a> Methods. 57 (4), 441-447 (2012).</li><li>Rigaut, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+generic+protein+purification+method+for+protein+complex+characterization+and+proteome+exploration.">A generic protein purification method for protein complex characterization and proteome exploration.</a> Nat Biotechnol. 17 (10), 1030-1032 (1999).</li><li>Puig, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Tandem+Affinity+Purification+(TAP)+Method:+A+General+Procedure+of+Protein+Complex+Purification.">The Tandem Affinity Purification (TAP) Method: A General Procedure of Protein Complex Purification.</a> Methods. 24 (3), 218-229 (2001).</li><li>Gavin, A. -. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+organization+of+the+yeast+proteome+by+systematic+analysis+of+protein+complexes.">Functional organization of the yeast proteome by systematic analysis of protein complexes.</a> Nature. 415 (6868), 141-147 (2002).</li><li>Tripathi, P., Carvallo, M., Hamilton, E. E., Preuss, S., Kay, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+B-BOX32+interacts+with+CONSTANS-LIKE3+to+regulate+flowering.">Arabidopsis B-BOX32 interacts with CONSTANS-LIKE3 to regulate flowering.</a> Proc Natl Acad Sci USA. 114 (1), 172-177 (2017).</li><li>Helfer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LUX+ARRHYTHMO+encodes+a+nighttime+repressor+of+circadian+gene+expression+in+the+Arabidopsis+core+clock.">LUX ARRHYTHMO encodes a nighttime repressor of circadian gene expression in the Arabidopsis core clock.</a> Curr Biol : CB. 21 (2), 126-133 (2011).</li><li>Pruneda-Paz, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-scale+resource+for+the+functional+characterization+of+Arabidopsis+transcription+factors.">A genome-scale resource for the functional characterization of Arabidopsis transcription factors.</a> Cell Rep. 8 (2), 622-632 (2014).</li><li>Wanke, D., Harter, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Plant+Regulatory+DNA+sequences+by+the+Yeast-One-Hybrid+Assay.">Analysis of Plant Regulatory DNA sequences by the Yeast-One-Hybrid Assay.</a> Methods Mol Biol. 479, 291-309 (2009).</li><li>Klein, P., Dietz, K. -. J. <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+DNA-Binding+Proteins+and+Protein-Protein+Interactions+by+Yeast+One-Hybrid+and+Yeast+Two-Hybrid+Screen.">Identification of DNA-Binding Proteins and Protein-Protein Interactions by Yeast One-Hybrid and Yeast Two-Hybrid Screen.</a> Methods Mol Biol. 639, 171-192 (2010).</li><li>Breton, G., Kay, S. A., Pruneda-Paz, J. L. <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+Arabidopsis+Transcriptional+Regulators+by+Yeast+One-Hybrid+Screens+Using+a+Transcription+Factor+ORFeome.">Identification of Arabidopsis Transcriptional Regulators by Yeast One-Hybrid Screens Using a Transcription Factor ORFeome.</a> Methods Mol Biol. 1398, 107-118 (2016).</li><li>Deplancke, B., Vermeirssen, V., Arda, H. E., Martinez, N. J., Walhout, A. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gateway-Compatible+Yeast+One-Hybrid+Screens.">Gateway-Compatible Yeast One-Hybrid Screens.</a> CSH Protoc. 2006 (5), (2006).</li><li>Deplancke, B., Dupuy, D., Vidal, M., Walhout, A. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gateway-compatible+yeast+one-hybrid+system.">A gateway-compatible yeast one-hybrid system.</a> Genome Res. 14 (10B), 2093-2101 (2004).</li></ol>

作者声明没有利益冲突。

现有的Y1H标准方法适用于鉴定与其DNA诱饵结合的单一蛋白质猎物。通过各种技术修改，现有系统已被用于定义转录调控网络。然而，TF已知作为涉及两种或更多种TF或蛋白质的复合物的一部分，只有一些TF能够结合DNA。仅具有蛋白质结合结构域并且缺乏与DNA结合能力的蛋白质或TF更可能在复合物中起作用，优选使用能够结合DNA的TF。使用传统Y1H的高通量屏幕偶尔会忽略这些生物重要的相互作用。因此...

<html>一般地，为了了解蛋白质-DNA相互作用，所述Y1H测定是在实验室设置1成功地使用的优选系统。基本的Y1H测定涉及两个组分:a）在编码报道蛋白的基因上游成功克隆了目标DNA的报道构建体;和b）将在感兴趣的TF和酵母转录激活结构域（AD）之间产生融合蛋白的表达构建体。感兴趣的DNA通常被称为"诱饵"，而融合蛋白被称为"猎物"。在过去几年中，已经开发了多种版本的Y1H测定法，以适应具体需求，具有自己的优点2 。可以成功实施现有的Y1H测定，以一次鉴定和验证一种蛋白质与其DNA诱饵的相互作用，但缺乏识别异二聚体或多聚体蛋白质 - DNA相互作用的能力。 "&gt;这里描述的方法是现有Y1H的修改版本，使研究人员能够同时研究与其靶DNA序列结合的多种蛋白质，该测定的目的是用激活结构域表达感兴趣的蛋白质（pDEST22: TF），并在酵母系统中存在和/或不存在相互作用的蛋白质配偶体（pDEST32ΔDBD-TF）的情况下评估与报告子融合的候选DNA区域的活化，该测定将允许我们确定这两个蛋白质是激活靶标所必需的，这是一种GATEWAY克隆兼容系统，因此可用于常规实验室设置，该方案基于直接转化，提供了酵母系统中不同TFs的共转化因此，验证蛋白质复合物与其可能的DNA靶标结合的体外分子和功能验证是一个有利的策略或任何系统。如串联亲和纯化（TAP）的方法目前的技术是昂贵的和劳动密集的，但允许大规模3，4，5蛋白hetrocomplexes的检测。这种修饰的酵母单杂交体可以在小规模的常规实验室设置中成功进行（ 图1 ），并且也具有成本效益。 Y1.5H测定可以促进社区的研究人员测试和验证他们的假设，以使用异源系统定义多聚体蛋白复合物与常见DNA靶标的作用。根据研究结果，该假设可以在优选的研究系统中在体内得到验证。最近，我们采用这种方法来定义TF的作用及其作用方式来调节拟南芥6的开花。

<table><tbody><tr><td>pDEST22 vector</td><td>Invitrogen</td><td>PQ1000101</td><td>Pro-Quest Two hybrid system kit</td></tr><tr><td>pDEST32delatDBD</td><td>Invitrogen</td><td>PQ1000102</td><td>Pro-Quest Two hybrid system kit; this vecor is modified. It is pDEST32 minus DNA binding Domain</td></tr><tr><td>pENTR/D-TOPO Cloning Kit</td><td>Invitrogen</td><td>K240020</td><td></td></tr><tr><td>YM4271 Strain</td><td>Clontech</td><td>K1603-1</td><td>MATCHMAKER One-Hybrid System</td></tr><tr><td>pEXP-AD502</td><td>Invitrogen</td><td>PQ1000101</td><td>Pro-Quest Two hybrid system kit</td></tr><tr><td>GATEWAY LR Clonase II enzyme mix</td><td>Invitrogen</td><td>11791020</td><td></td></tr><tr><td>YPDA media</td><td>Clontech</td><td>630410</td><td></td></tr><tr><td>SD-Agar</td><td>Clontech</td><td>630412</td><td></td></tr><tr><td>SD minimal media</td><td>Clontech</td><td>630411</td><td></td></tr><tr><td>Uracil DO Supplement</td><td>Clontech</td><td>630416</td><td></td></tr><tr><td>Tryptophan Do Supplement</td><td>Clontech</td><td>630413</td><td></td></tr><tr><td>Tris Base</td><td>Fisher Scientific</td><td>BP152-1</td><td></td></tr><tr><td>EDTA</td><td>Fisher Scientific</td><td>S311-500</td><td></td></tr><tr><td>LiAc</td><td>Sigma-Aldrich</td><td>L4158</td><td></td></tr><tr><td>Saplmon Sperm (10mg/ml)</td><td>Invitrogen</td><td>15632011</td><td></td></tr><tr><td>96 well round bottom Plate</td><td>Greiner bio-one</td><td>650101</td><td></td></tr><tr><td>PEG3350</td><td>Sigma-Aldrich</td><td>1546547</td><td></td></tr><tr><td>96-deep well block</td><td>USA Scientific</td><td>1896-2000</td><td></td></tr><tr><td>Sealable Foil</td><td>USA Scientific</td><td>2923-0110</td><td></td></tr><tr><td>Araseal</td><td>Excel Scientific</td><td>B-100</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Fisher Scientific</td><td>034461-100</td><td></td></tr><tr><td>ONPG</td><td>Sigma-Aldrich</td><td>73660</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>223484</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S3264</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S3139</td><td></td></tr><tr><td>KCL</td><td>Fisher Scientific</td><td>BP366-500</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>83266</td><td></td></tr><tr><td>HCL</td><td>Fisher Scientific</td><td>SA54-4</td><td></td></tr><tr><td>Drybath</td><td>Thermo Fischer</td><td></td><td></td></tr><tr><td>Voretx</td><td>Thermo Fischer</td><td></td><td></td></tr><tr><td>Centrifgue</td><td>Eppendorf</td><td>Centrifuge 5810R</td><td></td></tr><tr><td>Plate Reader</td><td>Molecular Device</td><td></td><td>SPECTRAMAX PLUS Microplate Spectrophotometer</td></tr><tr><td>Incubator</td><td>Thermo Fisher</td><td>Model No. 5250- 37 Degree, 6250-30 degrees</td><td></td></tr><tr><td>Shaker Incubator</td><td>New Brunswick</td><td></td><td></td></tr><tr><td>Water Bath</td><td>Thermo Fisher</td><td>IsoTemp 205</td><td></td></tr><tr><td>Puncher</td><td></td><td></td><td></td></tr><tr><td>50 ml Falocn</td><td>BD Falcon</td><td></td><td></td></tr><tr><td>Beaker</td><td>Nalgene</td><td></td><td></td></tr><tr><td>Flask</td><td>Nalgene</td><td></td><td></td></tr><tr><td>Petriplates (150mm)</td><td>Greiner bio-one</td><td></td><td></td></tr></tbody></table>

a modified yeast-one hybrid system for heteromeric protein complex-dna interaction studies

多年来，酵母单杂交测定已被证明是识别和验证蛋白质（如转录因子（TF））及其DNA靶标之间的物理相互作用的重要技术。本文提出的方法利用Y1H的基本概念，但进一步修改以研究和验证与其靶DNA结合的蛋白质复合物。因此，其被称为修饰的酵母单杂交（Y1.5H）测定。该测定是成本有效的，并且可以在常规实验室设置中容易地进行。 尽管使用异源系统，但是所描述的方法可能是用于在任何研究系统，特别是植物基因组学中测试和验证异源蛋白复合物与其用于功能基因组学的DNA靶标结合的有价值的工具。

用于共同转化酵母细胞中DNA区域与感兴趣蛋白质的一般平板安装程序（ 图2 ）。可以根据实验的需要和测试的DNA区域/片段的数量来修改平板设置。在方案的第5天之后， 如图3所示，可以看到良好生长和阳性的平板。在我们的研究中，从转录起始位点开始上游的2600bp的启动子区被分成6个区域或片段（FR 1-6） 6 ?...

Watch this Scientific Journal Video about A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies at JoVE.com

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

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

这里描述的修饰的酵母单杂交测定法是经典酵母单杂交（Y1H）测定法的扩展，以研究和验证异源蛋白复合物 - DNA异构体系中任何功能基因组学研究的相互作用。

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

a-modified-yeast-one-hybrid-system-for-heteromeric-protein-complex

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

11.3K 次观看.  University of Southern California. 这里描述的修饰的酵母单杂交测定法是经典酵母单杂交（Y1H）测定法的扩展，以研究和验证异源蛋白复合物 - DNA异构体系中任何功能基因组学研究的相互作用。 

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

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

Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and computational approaches. One such method is the yeast one-hybrid (Y1H) assay, in which interactions between TFs and DNA regions are tested in the milieu of the yeast nucleus using reporter genes. Y1H assays involve two components: a &#8216;DNA-bait&#8217; (e.g., promoters, enhancers, silencers, etc.) and a &#8216;TF-prey,&#8217; which can be screened for reporter gene activation. Most published protocols for performing Y1H screens are based on transforming TF-prey libraries or arrays into DNA-bait yeast strains. Here, we describe a pipeline, called enhanced Y1H (eY1H) assays, where TF-DNA interactions are interrogated by mating DNA-bait strains with an arrayed collection of TF-prey strains using a high density array (HDA) robotic platform that allows screening in a 1,536 colony format. This allows for a dramatic increase in throughput (60 DNA-bait sequences against &#62;1,000 TFs takes two weeks per researcher) and reproducibility. We illustrate the different types of expected results by testing human promoter sequences against an array of 1,086 human TFs, as well as examples of issues that can arise during screens and how to troubleshoot them.

Here, we present an enhanced yeast one-hybrid screening protocol to identify the transcription factors (TFs) that can bind to a human DNA region of interest. This method uses a high-throughput screening pipeline that can interrogate the binding of &#62;1,000 TFs in a single experiment.

强化酵母单杂交筛网识别与人 dna 序列结合的转录因子

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and ...

遗传学

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 ...

<meta charset="utf8"></head><body>通过酵母表面显示技术优化蛋白质功能

Protein Engineering by Yeast Surface Display

Protein engineering enables the improvement of existing functions of a given protein or the generation of novel functions. One of the most widely used and versatile tools in the protein engineering field is yeast surface display, where a pool of randomized proteins is expressed on the surface of yeast. The linkage of phenotype (e.g., binding of the yeast-displayed protein to the antigen of interest) and genotype (the plasmid encoding for the protein variant) enables selection of this library for desired properties and subsequent sequencing of enriched variants. By combining magnetic bead selection with flow cytometric sorting, protein variants with enhanced binding to a target antigen can be selected and enriched. Notably, in addition to affinity maturation, binding to a target can also be achieved without any initial binding affinity. Here, we provide a step-by-step protocol that covers all essential parts of a yeast surface display selection campaign and gives examples of typical yeast surface display results. We demonstrate that yeast surface display is a broadly applicable and robust method that can be established in any molecular biology laboratory with access to flow cytometry.

<meta charset="utf8"></head><body>通过 Yeast Surface Display 进行蛋白质工程

This protocol describes the essential steps for conducting yeast surface display selection campaigns to enrich protein variants binding to an antigen of interest.

通过 Yeast Surface Display 进行蛋白质工程

Protein engineering enables the improvement of existing functions of a given protein or the generation of novel functions. One of the most widely used and ...

生物工程

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 ...

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

摘要

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此视频中的章节

酵母 2-混合筛在批次中比较蛋白质相互作用

强化酵母单杂交筛网识别与人 dna 序列结合的转录因子

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

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

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

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

通过 Yeast Surface Display 进行蛋白质工程

通过 Yeast Surface Display 进行蛋白质工程

通过 Yeast Surface Display 进行蛋白质工程

遗传和生物化学方法