Name: transmembrane domain oligomerization propensity determined by toxr assay
Uploaded: 2011-05-26T14:00:00
Description: Watch this Scientific Journal Video about Transmembrane Domain Oligomerization Propensity determined by ToxR Assay at JoVE.com

我们感谢美国国立卫生研究院（1R21CA138373站起来，对这项工作的财政支持（SU2C）癌症。HY是感激，2009年亿利奖由美国癌症研究，从悉尼金梅尔基金会金梅尔学者奖协会癌症研究中心（SKF - 08 - 101），和美国国家科学基金会教师早期职业奖（NSF0954819）。

1。克隆的注意事项<ol><li> NheI和BamHI酶切位点和5&#39; -磷酸两侧的利益的战区导弹防御系统的商业化准备寡核苷酸可以连接到pTox7（我们实验室的一个碱基对的插入，修改后直接酶 BamHI酶切位点14）（图3）消化顺序用BamHI和NheI。寡核苷酸是一个例子所示： 5&#39;ctagcTMDSEQUENCEg3“ 3“gTMDSEQUENCEcctag5” </li></ol> TMD的顺序应该是12-24残基（短序列大概拉长矢量编码的疏?...

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ToxR转录报告基因检测是一个浅显的方式，以确定有oligomerize潜力的跨膜序列。由于内的细菌内膜发生的相互作用，这个实验绕过膜模仿的环境中学习系统的有效性相关的问题。这个实验，克隆到一个单一的质粒多个TMDS可以很容易地并行执行，并可以进行整个实验在96孔板格式，可用于高通量分析蛋白质序列的大量。一旦17已检测到的相互作用，可关键的功能残留审问突变分析，使所涉及?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> BamHI位的限制酶</td><td> Invitrogen公司</td><td> 15201023 </td><td> Invitrogen公司的酶被发现比其他供应商更有效率</td></tr><tr><td> NheI限制酶</td><td> Invitrogen公司</td><td> 15444011 </td><td> Invitrogen公司的酶被发现比其他供应商更有效率</td></tr><tr><td> 15 mL培养管</td><td> Fisher Scientific则</td><td> 14 - 956 - 1J </td><td></td></tr><tr><td> SoC媒体</td><td> Teknova </td><td> S0225 </td><td>合适的音量和高压灭菌消毒。 </td></tr><tr><td> LB培养基</td><td> Sigma - Aldrich公司</td><td> L7275 </td><td>合适的音量和高压灭菌消毒。 </td></tr><tr><td>氯霉素</td><td> Sigma - Aldrich公司</td><td> CO 3的 78 </td><td>存放在冰箱的库存为30毫克/毫升的溶液中乙醇</td></tr><tr><td>阿拉伯糖</td><td> Fluka公司</td><td> 10839 </td><td>存放在冷冻水（W / V）的2.5％股份的解决方案</td></tr><tr><td> NA 2 HPO 4 </td><td> Sigma - Aldrich公司</td><td> S9390 </td><td></td></tr><tr><td>的NaH 2 PO 4 </td><td> Sigma - Aldrich公司</td><td> S9638 </td><td></td></tr><tr><td>氯化钾</td><td> Mallinckrodt化学品</td><td> 6858-06 </td><td></td></tr><tr><td>硫酸镁4 .7 H 2 O </td><td> Sigma - Aldrich公司</td><td> 63138 </td><td></td></tr><tr><td>十二烷基硫酸钠（SDS） </td><td> Sigma - Aldrich公司</td><td> L6026 </td><td></td></tr><tr><td> 2 - 硝基苯β- D -半乳糖苷（ONPG） </td><td> Sigma - Aldrich公司</td><td> 73660 </td><td></td></tr><tr><td> Z -缓冲</td><td></td><td></td><td> 16.1克钠2 HPO 4 5.5克的NaH 2 PO 4 0.75克氯化钾 0.246克硫酸镁4 化妆至1 L，pH值7.0 </td></tr><tr><td> Z-buffer/chloroform </td><td></td><td></td><td> 200毫升的β-巯基乙醇，2 mL氯仿，补至20 mL，与Z -缓冲。涡1分钟，1分钟800转的离心机。做出新的每块板。 </td></tr><tr><td> Z-buffer/SDS </td><td></td><td></td><td> 160毫克SDS溶解于10 mL Z -缓冲</td></tr><tr><td> Z-buffer/ONPG </td><td></td><td></td><td> 40毫克ONPG于10 mL Z -缓冲。做出新的每块板</td></tr><tr><td> β-巯基乙醇</td><td> Calbiochem </td><td> 444203 </td><td></td></tr><tr><td>抗- MBP单克隆抗体（HRP标记） </td><td> NEB </td><td> E8038S </td><td></td></tr><tr><td>最小媒体与麦芽糖</td><td></td><td></td><td> 1个M9的盐，0.4％麦芽糖，1毫克/毫升硫胺素，2毫米硫酸镁 </td></tr><tr><td> 96孔平底板</td><td> Sarstedt </td><td> 83.1835.300 </td><td></td></tr><tr><td>板阅读器</td><td> Beckman Coulter公司</td><td> DTX880模检测器</td><td></td></tr><tr><td>水浴</td><td> VWRI </td><td> 89032-204 </td><td></td></tr><tr><td>摇床</td><td> FormaScientific </td><td></td><td></td></tr></tbody></table>

transmembrane domain oligomerization propensity determined by toxr assay

蛋白质跨膜结构域的过于简单化的看法只是在磷脂双层锚早已disproven。跨膜蛋白在许多情况下，已经发展了高度复杂的行动机制。1-3的一种方式在膜蛋白可以调节自己的结构和功能是疏水螺旋的直接和具体的联系，形成结构化的跨膜低聚物4,5近来工作的重点分布在膜环境优先氨基酸在水溶液和不同的分子间作用力，驱动蛋白协会。6,7然而，在跨膜结构域的蛋白质的分子识别研究仍落后于那些滞后相比水溶性地区。一个主要的障碍依然存在：尽管跨膜齐聚可以实现显着的特异性和亲和力，他们协会的8直接测量是具有挑战性的。可应用的整合膜蛋白的功能研究的传统方法阻碍了固有的不溶性考试序列。生物物理研究代表跨膜结构域的合成肽获得的见解可以提供有用的结构的洞察力。然而，洗涤剂胶束或在这些研究中用来模仿细胞膜的脂质体系统的生物相关性往往是质疑;做肽在这些条件下采取类似母语的结构和其功能的行为，真正体现内的原生膜的行动模式？为了研究天然磷脂双层的跨膜序列之间的相互作用，Langosch实验室开发ToxR转录记者分析。利益的跨膜结构域嵌合蛋白与麦芽糖结合蛋白的位置，周质和ToxR表示提供的一份报告齐聚水平（图1）。 在过去的十年中，几个其他团体（如Engelman，DeGrado，夏嘉曦）进一步优化和应用这ToxR记者分析。10-13各种ToxR检测已成为金标准，以测试在细胞膜蛋白质相互作用。我们在此展示一个典型的实验操作，在我们的实验室，主要如下Langosch开发协议中进行。这个普遍适用的方法是非常有用的跨膜结构域自我协会在大肠杆菌中的分析大肠杆菌 ，其中β-半乳糖苷酶的生产是用来评估TMD齐聚倾向。战区导弹防御系统，诱导二聚化后，ToxR结合造成的β-半乳糖苷酶的LacZ基因调节的 CTX发起人。 ONPG除了lyzed细胞是通过比色读数。生产的光吸收物种O - nitrophenolate（ONP）（图2）中的β-半乳糖苷酶结果的ONPG水解断裂。

Watch this Scientific Journal Video about Transmembrane Domain Oligomerization Propensity determined by ToxR Assay at JoVE.com

Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

一个高效的程序，以评估单通跨膜结构域的寡聚化倾向（TMDS）描述。融合到ToxR的战区导弹防御系统组成的嵌合蛋白在大肠杆菌记者应变中表达。 TMD诱导齐聚的原因ToxR，记者​​蛋白的转录和生产，半乳糖苷酶的激活二聚体。

跨膜结构域齐聚倾向ToxR含量决定

The oversimplified view of protein transmembrane domains as merely anchors in phospholipid bilayers has long since been disproven.  In many cases ...

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