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索取请求提交后。 MET25甲硫氨酸阻遏的启动子控制的FFL-GFP质粒的融合蛋白的表达。质粒转化到每一株被改编从Gietz 等使用的协议。人 ，1995年: <ol><li>离心25毫升Ø<html> f日志期细胞50毫升锥形管2分钟，在4,400 rpm的转速下，用500微升的双蒸H 2 0（DDH 2 O），弃上清。 </li><li>将细胞重新悬浮在250μl的TE /醋酸锂（的Tris-HCl的10mM，pH值= 7.5，1mM EDTA的溶液，0.1M乙酸锂）。搅拌20分钟，在30°C孵育没有。 </li><li> 50μl的感受态细胞转移到新的试管，以及载体DNA，用5毫升（50微克）和5μl（0.1-5微克）质粒DNA。添加300μl的PEG / TE /醋酸锂（相同的TE /醋酸锂加40％的聚乙二醇），此混合物中加入另外的30分钟，在30℃下孵育。 </li><li> 1/10体积（体积/体积）的DMSO（36微升）加入到该混合物中，并在42℃下热休克6分钟。 </li><li>混合物以6,000 rpm离心30秒，除去上清液。细胞重悬在100μl的无菌DDH 2 O和板细胞在缺乏尿嘧啶（SC-URA）的合成完整的媒体。 </li></ol> 2。 FFL-GFP诱导NT"&gt; FFL-GFP诱导，一旦细胞对数生长期，所以大部分的蛋白质，热休克前新，以避免随着时间的推移，由于衰老相关的细胞不足蛋白质，身患汇总。 <ol><li>第1天:孵育细胞在5毫升SC-市建局在30°C旋转O / N。 </li><li>第2天:测量外径600和5 SC-URA外径600〜0.2毫升接种新鲜文化。 </li><li>培养与旋转30°C，直到他们达到数生长期外径600〜0.8-1.0。 </li><li> 3分钟后，倾去上清液，重悬细胞沉淀在15毫升锥形管，在4,400 rpm转速离心细胞DDH 2 0 500微升;离心管中的传输解决方案。 </li><li>在6000转离心，弃上清。 </li><li>重悬细胞SC-URA-MET在5毫升。细胞应在30℃下孵育1小时，在这样的介质旋转</li><li>离心机细胞和重复洗的DDH 2 0丢弃上清。 </li><li> Resuspe第二细胞在5毫升SC-市建局媒体含有100微克/毫升，放线菌酮抑制蛋白的表达，并立即进行步骤3。 </li></ol> 3。酶法FFL-GFP恢复含量此法是一种定量的方法来确定在一个群体中的活性酶的水平。 <ol><li>这个实验的准备工作: <ol class="lroman"><li>测量每个样品外径600。 </li><li>准备所需的必要量的D-荧光素的基础上的样本数量和所需数量的复加油管约1200微升。 D-荧光素的终浓度为〜23微克每100μl的细胞。最初，它是溶解在水中的储备液浓度为7.5毫克/毫升，实验前稀释至455微克/毫升在SC。在图2中，重复三次，对每个样品进行了分析，得到的共2.4毫升D-荧光素被使用。 </li><li>程序酶标仪的闪光LUMI释光测定（BioTek的GEN5"发光闪光含量与注射"改编自其他工具按照制造商的说明） <ol class="lalpha"><li>至30℃的设定温度</li><li>设置钢板读者添加D-荧光素，摇匀，并读取每个单独。 </li><li>阅读发光（端点读取积分时间，5秒，180灵敏​​度电平） </li><li>恢复检测每个读数之间摇5分钟，延时20分钟，摇5分钟。 </li><li>免除50微升的D-荧光素从一个喷油器。 </li></ol></li></ol></li><li>后立即测量的细胞中添加介质中放线菌酮，阻止蛋白质的合成预热休克发光的。 <ol class="lroman"><li>每个样品100微升细胞转移成固体白色96孔板中所需的重复数。 </li><li>应包括控制水的空白，并含有空载体的细胞。 </li><li>开始读与SPE上面列出的cifications。 </li></ol></li><li>变性，的平板荧光灯-GFP FFL部分，在一个玻璃培养管中孵育5毫升培养在42℃下摇动25分钟。 </li><li>恢复化验发光读数立即启动细胞去除后，从孵化器，这是时间点零。测量发光上述相同的第一时间点和每30分钟90分钟。 </li><li>将正常化样品预热休克发光值已作调整的基础上外径600（外径600划分的预热冲击值）。 </li></ol> 4。荧光显微镜此法是一种半定量的方法来确定随着时间的推移在一个群体中的细胞聚集体溶解。 <ol><li>感应第2节中所描述的相同，步骤1-8的协议。 </li><li>同一时间点作为上述（ 第3项，第4步 ），但对于麦克风roscopy收集1毫升预热休克细胞，每个恢复的时间点，并孵育样品，于30℃培养管旋转。 </li><li>可视化: <ol class="lroman"><li> 1毫升的细胞以6,000 rpm离心30秒，除去上清液。 </li><li>重悬细胞沉淀中加入2μl双蒸2 O。 </li><li>混合2微升的细胞加2微升2％的低熔融琼脂糖幻灯片上，加盖玻片。为了获得一个单一的平面的细胞轻轻地按压手指在盖玻片的中心和旋转，直到盖玻片难以移动。 </li><li>细胞可视化使用100X油目标的荧光显微镜使用DIC可视化细胞和FITC过滤的可视化绿色荧光。 </li><li>每个菌株在各时间点的定量，以多张图片。图片字段应该包含〜15-30细胞实验后定量分析。 </li><li>要包含聚集的细胞百分比计算，算上50-100细胞总数除以包含聚集的细胞。 </li></ol></li></ol> 5。单细胞显微镜使用这种方法时，按照随着时间的推移在单个细胞中的单个聚集体的增溶。 <ol><li>诱导FFL-GFP的表达， 第2节中所描述的步骤1-8。 </li><li>热休克后，离心细胞15毫升锥形管，在4,400 rpm的转速为2分钟。 </li><li>洗涤细胞用500μl的水重悬在20微升双蒸2 O。 </li><li>对于每个应变，切出部分的SC-市建局的琼脂平板和一个5x5毫米使用表面接触培养皿，吸管4μL枪头，琼脂表面的细胞和周围蔓延。让我们站在〜1分钟。 </li><li>使用镊子，将琼脂立方体正面朝下放在一个〜55毫米的玻璃底菜0号，轻轻按下。 </li><li>可视化: <ol class="lroman"><li> 教I-III离子4。 </li><li>设置2-4重点每一株取决于密度的文化坐标。 </li><li>设置适当的范围（ - / +与0.5-1.0微米的切片厚度15微米）90分钟内每5分钟一个Z堆栈相机拍照。 </li></ol></li></ol>

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没有利益冲突的声明。

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>
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 <td>Olympus IX81-ZDC Confocal Inverted Microscope</td>
 <td>Olympus, Tokyo Japan</td>
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 <td></td>
 </tr>
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 <td>Lumitrac 200 white 96-well plates</td>
 <td>USA Scientific</td>
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 <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 ...

coupled-assays-for-monitoring-protein-refolding-saccharomyces

Research

JoVE Journal

Biology

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

10.2K 次观看.  University of Texas Medical School. 本文介绍了利用萤火虫荧光素-GFP融合蛋白进行调查在地体内蛋白质折叠酿酒酵母（Saccharomyces cerevisiae）。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。

视频: Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

剖析酿酒酵母 ASCI

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

科研

生物学

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

脂肪酸定量磷酸化刺激酿酒酵母</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

微阵列分析FOR酿酒酵母</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

囊性纤维化跨膜传导调节蛋白表达和纯化酿酒酵母

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

囊性纤维化跨膜电导调节蛋白表达纯化<em&gt;酿酒酵母</em

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

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell&#8217;s specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.

mRNA的衰变率的测量<em&gt;酿酒酵母</em&gt;使用<em&gt; rpb1-1</em&gt;株

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

化学遗传相互作用的快速鉴定<em&gt;酿酒酵母</em

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

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.