这项工作是由财政从荷兰国家研究学院结合，催化化学设计（NRSC-催化）控制的研究资助。 HY Steensma博士和GPH面包车赫斯登博士莱顿大学都承认他们与S的重组表达支持酵母 Nar1。

1.准备设置和解决方案<ol><li>制备含有100mM的Tris，250mM的NaCl和10％甘油在pH 8.5缓冲溶液。通过用氩气冲洗脱气缓冲。引入缓冲器成厌氧室中。 </li><li>划分的缓冲器中的两个部分。一个部分（缓冲液A）中添加1mM的DL-二硫苏糖醇（DTT，M R154.25克/摩尔）和1mM L-半胱氨酸。其它部分（缓冲液B）不包含还原剂。 </li><li>制备氧化剂和还原剂溶液。使1毫升溶液2，20，和200毫铁氰化钾（FIC，M R329.26克/摩尔）和连二亚硫酸钠（SD，M R174.11克/摩尔）在厌氧缓冲器B. </li><li>准备调停结构如下： <ol><li>添加160μM的每种以下的氧化还原介体和50mM的NaCl的缓冲B：N，N，N&#39;，N“ -四甲基-对- phenylendediamine（TMPD，M R237.17克/摩尔，E&#39;0 0.276 V）的，2,6-二氯酚靛酚（DCIP，M R290.08克/摩尔，E&#39;0 0.217 V），吩嗪ethosulphate（PES，M R334.4克/摩尔，E&#39;0 0.055 V），亚甲蓝（Mr为319.85克/摩尔，E&#39; 0 0.011 V）中，试卤灵（Mr为235.17克/摩尔，E&#39;0 -0.051 V）中，Indigodisulphonate（Mr为466.35克/摩尔，E&#39;0 -0.125 V），2-羟基-1,4- naphtaquinone（ Mr为174.15克/摩尔，E&#39;0 -0.145 V），蒽醌-2-磺酸（Mr为328.28克/摩尔，E&#39;0 -0.225 V），Phenosafranin（Mr为322.8克/摩尔，E&#39;0 -0.252 V），番红O（Mr为350.84克/摩尔，E&#39;0 -0.280 V），中性红（Mr为288.8克/摩尔，E&#39;0 -0.340 V），苯紫精（Mr为409.4克/摩尔，E “0 -0.350 V）和甲基紫精（M R257.16克/摩尔，E&#39;0 -0.440 V）。 </li><li>包裹在铝箔的瓶子，以保护介期从光​​。 </li></ol></li><li>制备10毫克/毫升S。酵母链球菌标记Nar1蛋白（181微米，M R 55.2 kDa的）的缓冲A.快车S.酵母 Nar1在E.大肠杆菌作为链球菌标记蛋白使用杨pET24a表达载体中。采用链球菌-tactin层析介质（IBA）纯化该蛋白质在厌氧室中和纯化使用蛋白纯化系统如AKTA净化器（GE Healthcare）上。 </li><li>准备氧化还原滴定设置在厌氧室如下： <ol><li>连接一个Ag / AgCl参考电极和铂丝电极的滴定容器中。两个电极连接到可从毫伏测量多达五伏特计</li><li>插入一个小的搅拌棒。 </li><li>放置磁性搅拌器上方滴定容器中。 </li><li>填用缓冲液B，介体混合溶液和蛋白质溶液至高达2毫升， 即以最终浓度72μM的蛋白质和38μM每个滴定管调解员。注意：取决于蛋白质浓度和所需的介体浓度下，三种溶液的量应调整​​。 </li><li>搅拌30分钟，接通电压表。 </li></ol></li><li>准备10清洁石英玻璃EPR管。引入管在厌氧室中。 </li><li>填写小（ 大约 200ml）中与液氮液氮杜瓦。 </li></ol> 2.进行氧化还原滴定<ol><li>等到溶液的电位是稳定的。注意：在实践中，这意味着潜在的漂移小于每分钟1毫伏。 </li><li>记下的电压表给出的潜力。 </li><li>绘制的200微升第一个样品，注入的EPR管。 </li><li>盖上管用橡胶塞，并从厌氧室中取出试管。 </li><li>冻结管在液氮如下：小心！这必须慢慢地进行，否则该管将打破。 <ol><li>首先插入吨IP的管中的液氮。 </li><li>等到嘶嘶声可以听到。注意：这可能需要长达10秒。 </li><li>慢慢插入管的其余部分，使得样品部分被完全淹没。 </li></ol></li><li>加一个小的等分试样1-2微升2mM的SD溶液在滴定容器中的溶液，以降低该溶液的电位-0.6V。 注意：如果超过10微升已被添加，切换至20mM的溶液。因为它是非常困难的泊至一个特定的潜在价值，记录在电压表的实际值被记录下来，并用来构造滴定曲线。 </li><li>按照步骤2.3-2.5描述抽取样本。 </li><li>记下的电压表给出的实际潜力。 </li><li>添加氧化剂，直到潜在的增加了大约 50毫伏。开始加入小等分试样1-2微升2mM的FIC溶液。如果超过10微升已被添加，切换至20mM的溶液。 </li><li>记下豪瓦特得多的解决方案已被添加。注意：这是必要的，以校正在滴定过程中样品稀释。 </li><li>在步骤2.3-2.5描述抽取样本。 </li><li>记下的电压表给出的实际潜力。 </li><li>重复步骤2.9和2.12，直到潜在的范围从-0.6 V至0.2 V已覆盖全部10 EPR管200微升充满每一个。 注意：与每个撤出体积减小电极的定位可需要调整与未经搅拌的干扰的溶液有良好的接触。它可能是难以测量的最后一个样本的撤出，因此该样品的电势可以是较不准确后的潜力。 </li><li>任选地，储存冷冻EPR样品在液氮中直至EPR测量可以发生。 </li></ol> 3.测量滴定样品采用EPR谱和数据分析<ol><li>不同的记录EPR谱滴定样品如下： <ol><li>使用高真空泵撤离石英低温恒温器，以&lt;10 -5杆。 </li><li>接通EPR磁体的水冷却。开的干燥空气通过EPR腔的流动。 </li><li>接通电源，以在磁铁和EPR的计算机。 </li><li>运行频率校准程序。设置EPR测定参数。低温恒温器连接到液氦杜瓦。冷却腔，以9-16 K. </li><li>引入腔中的EPR样品。记录的EPR谱。 </li></ol></li><li>识别不同的EPR种类和使用的最佳光谱来量化的信号。实现的EPR定量由光谱和比较的外铜标准溶液光谱的双重积分的10mM 硫酸铜 / 10mm以上的HCl / 2M的的NaClO 4 8 </li><li>使滴定曲线如下： <ol><li>绘制的EPR信号幅度在g-值是特征为第磁种对潜在的利益。 </li><li>通过使用EPR定量来自步骤3.2改变的EPR信号幅度比例为自旋/分子尺度。 </li><li>校正每个样品为已发生因氧化或还原剂的添加，该稀释的信号振幅。 </li><li>校正为Ag / AgCl参考电极的中点电位的电位。 注意：Ag / AgCl参考电极（用饱和的KCl）具有0.194 V相对于标准氢电极（SHE），在25℃的中间电位。因此，如果在电压表的电位读出大约 -0.6 V时，参照SHE电位为-0.4 V. </li></ol></li><li>该信号适合于能斯特方程如下： 对于一个物种，是顺磁性的还原态： <img alt="式（1）" fo:content-width="2in" src="/files/ftp_upload/51611/51611eq1.jpg" width="200" /> 对于一个物种，是顺在oxidized状态： <img alt="公式2" fo:content-width="2in" src="/files/ftp_upload/51611/51611eq2.jpg" width="200" /> 对于一个物种，是顺处于中间状态： <img alt="公式3" fo:content-width="2.2in" src="/files/ftp_upload/51611/51611eq3.jpg" width="220" /> 这些方程是有效的，其中一个电子转移的氧化还原反应，因此n = 1。 E =潜力伏 ËM =在伏中点电位 F =法拉第常数= 96,480·C·摩尔-1 R =气体常数= 8.314·J·K -1·摩尔-1 T =温度在开尔文</li></ol>

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作者什么都没有透露。

该系列13的氧化还原介体允许从-0.45至+ 0.3V的SHE相比（ 表1和图3）的氧化还原平衡中，在范围内的溶液。井的上方和下方，这些电位所有介体或者完全降低或完全被氧化，因而没有进一步平衡与​​蛋白质的氧化还原活性中心可以发生。这是一个重要的概念，如在文献中有时氧化还原滴定与只有两个或三个介质进行的，只允许一个窄的范围内被测量11。这种氧化还?...

令人吃惊的是最基本的化学过程，在这个星球上，光合作用，固氮和呼吸维持生命，是由含有多种有机和无机氧化还原辅助因子大蛋白复合物催化。据估计，所有蛋白质的约30％包含一种或多种金属辅因子。1,2-识别和表征所述氧化还原辅助因子可以使用直接电化学（ 例如 ，蛋白质膜伏安）或氧化还原滴定来确定。这两种技术都在他们的先进性和适用性的互补性。伏安法提供快速测定中点电位和辅因子的电子转移动力学，可以与电极表面反应的3,4通常这可以很好地用于电子转移蛋白，如细 ​​胞色素C或铁氧还蛋白。它有时适用于已固定于电极表面更复杂的蛋白质。的详细知识在蛋白辅因子的性质必须是可用的，因为伏安不会给对辅因子的身份的任何直接信息。氧化还原滴定更费力执行，并要求毫克的量的蛋白质。然而，它们提供的中点电位和辅因子的身份的信息。5此外，在一个单一的滴定中的蛋白质的多个辅因子可以被监视。 氧化还原滴定的原则是，氧化还原活性蛋白质或酶进行化学还原或氧化。为了确保该辅因子与还原剂或氧化剂的氧化还原介体被用于反应。这些氧化还原媒介剂也发生反应的电极，以使溶液的电位可以被测量。该介质充当氧化还原缓冲液中的蛋白质和电极的辅因子之间平衡。后化学已达平衡的电位到期望值，样品被吸入，并迅速在液氮中冷冻，以AWAIT进一步分析光谱技术。因为它可用于定量测定顺磁性金属中心，或有机基团EPR谱是在这方面是特别有用的。 氧化还原滴定可以在两个方向进行：从低到高或由高向低中点电位。该选择取决于所研究的辅助因子的稳定性。往往是明智的，开始在低电位，并添加氧化剂，以逐步增加的潜力。这就是所谓的氧化滴定并在这里被描述。这里，我们表明含蛋白质Nar1来自酿酒酵母的铁-硫簇的氧化还原滴定。这种蛋白参与，有可能作为支架蛋白，在细胞内铁硫簇生物合成机械（CIA途径）。6,7

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Reference electrode (Ag/AgCl)</td>
			<td style="width:101px;">Radiometer</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chemicals</td>
			<td style="width:101px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EPR spectrometer</td>
			<td style="width:101px;">Bruker</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">N,N,N&#8217;,N&#8217;-tetramethyl-p-phenylendediamine (TMPD) &#183;(HCl)2</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">637-01-4</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">2,6-dichlorophenol indophenol (DCIP), sodium salt</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">620-45-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phenazine ethosulfate (PES)</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">10510-77-7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methylene blue</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">122965-43-9</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Resorufin, sodium salt</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">34994-50-8</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Indigodisulfonate (indigo carmine)</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">860-22-0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-hydroxy-1,4-naphtaquinone</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">83-72-7</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Anthraquinone-2-sulfonate Na+ H2O</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">153277-35-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phenosafranin</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">81-93-6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Safranin O</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">477-73-6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Neutral red</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">553-24-2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Benzyl viologen</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">1102-19-8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methyl viologen</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">1910-42-5</td>
		</tr>
	</tbody>
</table>

epr monitored redox titration of the cofactors of saccharomyces cerevisiae nar1

电子顺磁共振（EPR）监测氧化还原滴定是一个功能强大的方法来确定的辅助因子的中点电位中的蛋白质，并确定和量化的辅因子在其可检测的氧化还原状态。 该技术是互补的直接电化学（伏安）接近，因为它不提供对电子传输速率的信息，但是并正在研究建立在蛋白质的辅助因子的身份和氧化还原状态。该技术广泛适用于含有电子顺磁共振（EPR）检测辅因子的任何蛋白质。 一个典型的滴定需要2 ml蛋白质具有在1-100微米的范围内的辅因子浓度。该蛋白质以泊样品在一定的电势滴定用化学还原剂（连二亚硫酸钠）或氧化剂（铁氰化钾）。铂丝和一个Ag / AgCl参考电极连接到伏特计来测量蛋白质溶液的电位。一组13种不同的氧化还原介体时所使用的蛋白质的氧化还原辅因子和电极之间达到平衡。样品被绘制在不同的电位和电子顺磁共振光谱特性为在该蛋白质的不同的氧化还原辅因子，被测量。信号强度与样品潜在的情节是使用能斯特方程以确定该辅因子的中点电位进行分析。

含蛋白质Nar1来自酿酒酵母的铁-硫簇的氧化还原滴定导致三种不同的辅助因子的鉴定：Nar1：一个[3FE-4S]簇，一个[4FE-4S]簇和单核铁中心（ 图1） 。 EPR的信号的特征在于它们的G-值：对于[3FE-4S] + Gž2.01克2.00 交叉 （ 图1B）;对于[4FE-4S] +，Gž2.02克Ÿ1.93，G X 1.82（ 图C），和单核的Fe 3+克4.3和9.7（ ?...

Watch this Scientific Journal Video about EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1 at JoVE.com

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

The goal of this protocol is to use electron paramagnetic resonance (EPR) monitored redox titrations to identify different cofactors of Saccharomyces cerevisiae Nar1. Redox titrations offer a very robust way to obtain midpoint potentials of different redox active cofactors in enzymes and proteins.

EPR监控的辅助因子氧化还原滴定<em&gt;酿酒酵母</em&gt; Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to ...

epr-monitored-redox-titration-cofactors-saccharomyces-cerevisiae

Research

JoVE Journal

Biology

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

13.4K 次观看.  Delft University of Technology. The goal of this protocol is to use electron paramagnetic resonance (EPR) monitored redox titrations to identify different cofactors of Saccharomyces cerevisiae Nar1. Redox titrations offer a very robust way to obtain midpoint potentials of different redox active cofactors in enzymes and proteins.

视频: EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

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 ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

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 ...

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.