Name: coupled assays for monitoring protein refolding in saccharomyces cerevisiae
Uploaded: 2013-07-09T12:00:00
Description: Watch this Scientific Journal Video about Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae at JoVE.com

这项工作是支持由授予来自美国国立卫生研究院（NIGMS-074696）锦和美国社会的微生物罗伯特D.沃特金斯研究生研究奖学金JLA我们也感谢在多伦多大学的约翰·格洛弗提供的FFL ，GFP源质粒。

1。含FFL-GFP质粒菌株建设在这项研究中， 酿酒酵母菌株BY4741（MAT 一个 his3Δ1，leu2Δ0，met15Δ0，ura3Δ0）一起使用从酵母基因敲除集合（打开生物系统/ Thermo Scientific的）一个的HSP104缺失菌株。删除Hsp104特异性抗体免疫印迹分析证实。 FFL-GFP表达p426MET25 FFL-GFP，构建一个基于LEU2源质粒格洛弗实验室获得多伦多大学17索取?...

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A., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+disaggregation+mediated+by+heat-shock+protein+Hsp104.">Protein disaggregation mediated by heat-shock protein Hsp104.</a> Nature. 372, 475-478 (1994).</li><li>Penna, T. C., Ishii, M., Junior, A. P., Cholewa, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+stability+of+recombinant+green+fluorescent+protein+(GFPuv)+at+various+pH+values.">Thermal stability of recombinant green fluorescent protein (GFPuv) at various pH values.</a> Appl. Biochem. Biotechnol. , 113-116 (2004).</li><li>Seliger, H. H., Buck, J. B., Fastie, W. G., McElroy, W. 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在这篇文章中的模型蛋白FFL-GFP是用来显示该的酵母disaggregase，HSP104有助于蛋白质重新溶解和维修。的酶活分析和显微镜差异讯问的相同的底物蛋白的状态来确定的复性效率和产量。酶的恢复检测的结果表明，不仅是在的突变株hsp104D效率低下的最大恢复，但在突变株（ 图2）展开应力的初始幅度更大。分析还显示，虽然出现hsp104D细胞试图修复，他们无法重新折叠蛋白尽快...

在人类神经退行性疾病包括阿尔茨海默氏症，帕金森氏病和亨廷顿病已与蛋白质错误折叠和聚合10。细胞采用分子伴侣防止动能捕获细胞的蛋白质错误折叠的无效结构6。分子伴侣参与细胞内的复杂的相互作用网络，但它是不完全的理解这些相互作​​用的总和是如何有助于有机体proteostasis。负责为广大胞浆蛋白折叠的主要伴侣之一是70 kD的热休克蛋白（HSP70）家族19。它已经表明，在酵母中的Hsp70减少损失新生的异源表达的平板荧光灯能够折叠和重折叠，鸟氨酸transcarbamoylase，内源性蛋白7 在体内 18。能够分析近实时分辨率折叠方便了解更多的细胞因子续ribute这HSP70依赖的过程。此外，可能不完全依赖于这些蛋白质有助于折叠/折叠反应，所以实验必须敏感，可以探测动力学和效率的大，小的变化。 酵母细胞disaggregase，HSP104，在修复聚合错误折叠的蛋白质中起着至关重要的作用。 Hsp104同源物已确定在真菌和植物，这个家庭似乎是在后生动物缺席。已经提出了其他分子伴侣，如HSP110家族的一些已知的Hsp104在哺乳动物中的活动16执行。 HSP104为AAA +，六聚体蛋白复合物的功能在改造蛋白质聚集酵母，折叠和维修13。 HSP104，沿与Hsp70的酵母，SSA1，和酵母HSP40，Ydj1为，是必需的恢复变性平板荧光灯在酵母细胞中5星，8。小的热休克蛋白，HSP26，也被证明是地磁红的Hsp104介导分解FFL 2。 FFL是一个双结构域的蛋白结合的活性位点的构象变化，需要ATP和氧，脱羧后的衬底荧光素释放氧化萤光素，二氧化碳（CO 2），腺苷一磷酸（AMP），和光11，在基板9，3。市售FFL基板，D-荧光素，使用光度计15，可以检测到550-570纳米之间的光发射的结果。 FFL是极其敏感变性化学或热处理和聚合后迅速展开。当暴露在温度39-45°C之间的，FFL可逆展开和灭活12。相比之下，GFP及其衍生物具有很高的耐应力14蛋白质变性。因此，这两种蛋白质的融合使得FFL充当实验能够瞄准功能ĝ的不稳定部分FP细胞内的存款，可以用荧光显微镜可视化人口和单细胞水平。酶法测定半自动化加上自动显微镜的多模板读数器中的应用允许前所未有的复性反应的动力学和产量的同时评估。此外，轻便的分子遗传学模型真核生物酿酒酵母（Saccharomyces cerevisiae）允许的蛋白质的质量控制网络的机会，发现为基础的方法，以确定新的播放器，促进细胞的应激反应和proteostasis两个精确操纵。 在这项研究中，野生型（WT）和的HSP104缺失株FFL-GFP表达热休克蛋白变性。 FFL-GFP复性进行监测，通过酶法测定和代理读数显微镜维修表达蛋白质组过一个恢复时间当然。当野生型细胞相比，我们表明êHSP104折叠FFL-GFP突变株〜60％的效率较低，支持以前的研究结果，建立HSP104的激活变性FFL 2的一个角色。

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagents</td>
 </tr>
 <tr>
 <td>Synergy MX Microplate Reader</td>
 <td>BioTek</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Olympus IX81-ZDC Confocal Inverted Microscope</td>
 <td>Olympus, Tokyo Japan</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Lumitrac 200 white 96-well plates</td>
 <td>USA Scientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>SlideBook 5.0 digital microscopy software</td>
 <td>Intelligent Imaging Innovations, Inc. Denver, CO, USA</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Tris Base</td>
 <td>Fisher</td>
 <td>BP152-1</td>
 <td></td>
 </tr>
 <tr>
 <td>(LiAc) Lithium Acetate</td>
 <td>Sigma</td>
 <td>L6883</td>
 <td></td>
 </tr>
 <tr>
 <td>PEG (polyethelene glycol)</td>
 <td>Fisher</td>
 <td>BP233-1</td>
 <td></td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Fisher</td>
 <td>BP120-1</td>
 <td></td>
 </tr>
 <tr>
 <td>DMSO</td>
 <td>Malinckrodt</td>
 <td>4948</td>
 <td></td>
 </tr>
 <tr>
 <td>SC</td>
 <td>Sunrise</td>
 <td>1300-030</td>
 <td></td>
 </tr>
 <tr>
 <td>SC-URA</td>
 <td>Sunrise</td>
 <td>1306-030</td>
 <td></td>
 </tr>
 <tr>
 <td>cycloheximide</td>
 <td>Acros Organics</td>
 <td>357420050</td>
 <td></td>
 </tr>
 <tr>
 <td>D-luciferin</td>
 <td>Sigma</td>
 <td>L9504</td>
 <td></td>
 </tr>
 <tr>
 <td>low melt agarose</td>
 <td>NuSieve</td>
 <td>50082</td>
 <td></td>
 </tr>
 <tr>
 <td>immersion oil</td>
 <td>Cargille</td>
 <td>16484</td>
 <td></td>
 </tr>
 </tbody>
</table>

coupled assays for monitoring protein refolding in saccharomyces cerevisiae

proteostasis，定义为蛋白质折叠/生物合成的组合过程中，复性/维修，和退化，必须维护，以避免有害的后果1细胞是一个微妙的平衡。外部或内部因素破坏这种平衡可以导致蛋白质聚集，毒性和细胞死亡。对于人类来说，这是一个主要因素与神经退行性疾病相关的症状，如亨廷顿氏，帕金森氏症和阿尔茨海默氏病10。因此，它是必不可少的蛋白质参与维护proteostasis的被确定为了开发这些使人衰弱的疾病的治疗方法。本文中所描述的技术用于监测体内蛋白质折叠使用的模型蛋白萤火虫荧光素酶，绿色荧光蛋白（FFL-GFP）融合，以接近实时的时间分辨率。 FFL-GFP是一个独特的模型嵌合的蛋白质，FFL部分是极其敏感的应激诱导米isfolding和聚合，失活的酶12。监测使用酶法测定荧光素酶活性，在GFP部分提供了一种方法，可视化的水溶性或凝集的平板荧光灯用自动化显微镜。这些耦合的方法包括两个平行和技术上独立的方法来分析应力后一种酶复性和功能重新。恢复活性，可直接与分类和重溶动力学，以便更好地了解蛋白质的质量控制因素，如蛋白质伴侣协作来执行这些功能。此外，基因的缺失或突变可用于测试特定的蛋白质或蛋白质亚基的贡献，这个过程。在这篇文章中，我们研究的贡献HSP104的蛋白质disaggregase的13，被称为折叠系统5与Hsp40/70/nucleotide的交换因子（NEF）的合作伙伴，蛋白质折叠，来验证这种方法。

酵母是依赖于disaggregase，HSP104高效地热变性的蛋白质复性。 FFL-GFP的活动进行了监测热休克25分钟后，使用发光闪光检测图1。这个自动化的测定如图2所示的结果揭示了超过90分钟，最终导致了一个野生型细胞&gt; 80％恢复的活动逐步增加。该hsp104Δ应变恢复原有活性的19％，在同一时间帧。此外，初始失活程度远远高于伴侣突变株（26％活动在WT和11％hsp104Δ），...

Watch this Scientific Journal Video about Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae at JoVE.com

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

本文介绍了利用萤火虫荧光素-GFP融合蛋白进行调查<em&gt;在地体内</em&gt;蛋白质折叠<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em&gt;。使用这种试剂，可同时监控一个模型的热变性蛋白复性，通过荧光显微镜和酶法测定蛋白质的质量控制中，以探测作用的proteostasis网络组件。

耦合检测监控蛋白复性<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

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Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

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Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

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Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

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Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

定向进化法<em&gt;酿酒酵母</em&gt;：突变体库的创建和筛选

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...