这项工作是通过其CFTR三维结构协会由美国囊性纤维化基金会（CFF）。 TR是由英国的CF信托助学金，并通过数控英国BBSRC助学金。我们承认我们的同事在CFF CFTR三维结构财团的帮助和咨询，并为密码子优化的鸡CFTR序列和纯化标签的设计。

缓冲区1。准备<ol><li>使蛋白酶抑制剂的100倍的股票（PI）的鸡尾酒溶解96毫克AEBSF，3.5毫克胰凝乳蛋白酶抑制剂，10毫克E64，16.5毫克亮肽素，16.5毫克胃酶抑素，348毫克的PMSF，和4毫克苯丁抑制素在20毫升DMSO中。生产1毫升分装并储存于-20°C。使100倍的股票苄脒的，溶解720毫克的20ml超纯水（双蒸2 O）和1毫升的等分试样储存于-20℃。这个量是足够的纯化1。在所有的缓冲区，PI和脒库存用在1:100稀释。 </li><li>准备&#39;MPIB&#39;（0.3摩尔Tris pH值8，0.3M蔗糖，2mM的DTT）和&#39;CFTR&#39;（50毫摩尔Tris pH值8，20％（V / V）甘油，1mM DTT）的缓冲液和寒意至4℃ 。使用前，加入蛋白酶抑制剂鸡尾酒1:100，并根据MPIB量1:100苯脒用于重悬细胞沉淀（ 例如 ，使用3.5毫升PI和3.5ml苯脒在350毫升MPIB总体积）。 </li><li>制造Solubilization缓冲区。溶血磷脂酰甘油-14（LPG）的溶解缓冲液（50mM的Tris pH值8，10％（V / V）甘油，50 mM氯化钠，1mM的DTT，蛋白酶抑制剂（PIs）和4％（重量/体积）LPG）和十二烷基麦芽糖苷（DDM）的溶解缓冲液（50mM的Tris pH值8，20％（V / V）甘油，1 M氯化钠，1mM的DTT，蛋白酶抑制剂，4％（重量/体积）DDM）。缓冲区可以在超声波仪浴（35 W，40千赫）进行超声处理，以协助洗涤剂的分散，但要避免涡旋混合物，因为这会产生气泡。寒气至4℃后方可使用。 </li><li>用于液化石油气纯化CFTR纯化缓冲区的50mM Tris，10％（V / V）甘油，50 mM氯化钠，1mM的DTT，0.1％（重量/体积）LPG-14和蛋白酶抑制剂。准备350毫升这个缓冲区，和150毫升的相同缓冲液加1米咪唑。 pH调节两个缓冲器的8个。 </li><li>缓冲器用于纯化在DDM组成的50mM Tris pH值8，20％（V / V）甘油，1 M氯化钠，1mM的DTT，0.1％（重量/体积）的DDM。准备350毫升此缓冲液中，和150毫升的相同缓冲液加1M的咪唑乐。 pH调节两个缓冲器的8个。 </li><li>对于含有液化石油气凝胶渗透色谱法（GPC）缓冲液，制备的50mM Tris pH值8，10％（V / V）甘油，50 mM氯化钠，1mM的DTT，0.05％（重量/体积）LPG-14。用于GPC使用DDM制备的50mM Tris pH值8，20％（V / V）甘油，1 M氯化钠，1mM的DTT，0.1％（重量/体积）DDM的缓冲器。所有的缓冲液和双蒸2 O的GPC柱使用应该被过滤（0.2微米的过滤器），并在使用前脱气。 </li><li> SDS-PAGE样品缓冲液（2×工作浓度）：50mM的Tris-盐酸pH值7.6，5％（V / V）甘油，5mM的EDTA，0.02％（重量/体积）溴酚蓝。使700μL分装和储存在-20°C。在使用前，加入200微升的20％（重量/体积）十二烷基硫酸钠（SDS）和100μl的新鲜的0.5 M DTT。加载在凝胶上之前孵育至少10分钟，样品在室温下进行。不要加热;这将变性的GFP和可能导致的CFTR来聚合。 </li><li>为了使脂质股票的重建，化解E的4:1（W / W）的混合物大肠杆菌的脂类在氯仿和甲醇ð胆固醇（2:1 V / V），并在干燥N 2下的气体2小时的玻璃小瓶中，以形成脂质膜。添加GPC缓冲液（无NaCl）中，以40毫克/毫升的脂质浓度，并使用重复的涡旋和超声处理（35瓦，40千赫），以澄清溶液。 </li><li>为ATP酶测定法，通过溶解SCH28080至1mM在DMSO中，硫氰酸钠，以在DDH 2 O 1 M和寡到2.5毫米的100％（体积/体积）乙醇制备100倍的股票ATP酶的抑制剂。在储存分装于-20°C。 100毫升的ATP酶的缓冲液，用50mM的Tris pH值7.4，150mM的NH 4 Cl等 ，5 mM的MgSO 4干燥和0.02％（重量/体积）的NaN 3。这可以在室温下保存，并用于多种测定法。准备一个5毫米的ATP库存使用并保持在冰上前夕。 （NB使用的Na 2 ATP，以防止过 ​​度的背景信号从磷酸盐中测定）。准备SDS一站式解决方案（12％（W / V）SDS在DDH 2 O）。 </li><li>对于Chifflet检测准备缓冲液A（3％（W/ v）的抗坏血酸，0.5％（重量/体积）钼酸铵，0.5M HCl中）使用前立即和缓冲液B（2％（重量/体积）柠檬酸钠，2％（重量/体积）偏高碘酸钠砷，2％ （V / V）乙酸）。 </li></ol>酵母微粒体2的分离<ol><li> 酿酒酵母表达鸡CFTR的作为奥赖恩等人成长。（2012）14。从20升发酵在两等份在-80℃下储存材料长达6个月。 </li><li>解冻细胞的一个等份快速重悬在3ml冷MPIB每克细胞。 </li><li>扰乱中使用的425-600微米直径的玻璃珠的珠磨机细胞。用细胞破碎五1分钟周期由1分钟的休息时间分开。 （其余的期间是必要的，以确保细胞破坏过程中不进行加热。） </li><li>由1毫升样品从珠磨机的细胞裂解物离心监测细胞破碎。离心（12,000×g离心，4℃，5分钟）在台式离心泵ifuge。稀释上清液到1:50与MPIB在比色皿，测量380。如果A 380&gt; 0.1，或已经停止增长，尽管经过多次珠跳动周期，请执行以下步骤。如果没有，重复2.3-2.4。 </li><li>离心的总细胞裂解物（12,000× 克，4℃，20分钟）。保留上清液。弃去沉淀（含完整的细胞和线粒体），但如果有关于电池单电池破损的效率有任何疑问（见2.4），则保留该粒料也。 </li><li>从以前的步骤（200,000×g离心，4℃，1.5小时）离心上清液。弃上清，重悬在CFTR的缓冲区中的沉淀微粒体膜。如果微粒是供使用DDM纯化，补充CFTR缓冲用1M NaCl洗涤。 </li><li>重复该再悬浮的膜级分的离心分离（100,000× 克，4℃，1小时），并弃去上清液。 </li><li>悬浮的微粒沉淀的CFTR的缓冲区最小体积（终体积5-15毫升，总微粒体蛋白70-200毫克）。 Bradford测定法可用于确定微粒体蛋白25的总浓度。此外，膜的荧光发射光谱应测定（激发= 485 nm，发射= 500〜600纳米），并应具有明显的GFP荧光峰值（最大为512纳米）。 CFTR可以在SDS-PAGE凝胶上，GFP荧光的条件下扫描（ 图1）进行特异性检测。 </li><li>闪冻通过投入液氮内使之重新悬浮微粒，并存储于-80℃，或继续执行步骤3。 </li></ol>微粒3。溶<ol><li>如果冻结，立即在水浴中设定为10℃，使用前解冻微粒</li><li>对于膜的溶解，稀释的有关​​溶解缓冲液（步骤1.3）中加入等体积的微粒，以得到最终洗涤剂浓度2％（重量/体积）和微粒体蛋白浓度为5毫克/毫升。 1小时，在4℃搅拌下（管旋转体）孵育此混合物。保留200μL进行分析。 </li><li>离心混合物（100,000×g离心，4℃，45分钟）。除去含有溶解的膜蛋白的上清液，通过它通过0.45μm的注射器过滤器和储存在冰上。测量上清液的荧光（如步骤2.8）。 </li><li>悬浮在1％的不溶部分（重量/体积）SDS溶液至体积等于可溶部分。测量的荧光在该馏分中，并保留50μl的用于SDS-PAGE分析的等分试样。 </li></ol> 4，镍亲和CFTR的分离纯化<ol><li>连接两个5毫升镍琼脂糖凝胶柱串联。用2倍柱体积（CV）的洗涤的20％（V / V）乙醇，接着2 CV双蒸2 O，然后用2 CV的溶解缓冲液洗柱（步骤1.4-1.5），含有1M咪唑。 2简历solubilizat的重复离子缓冲缺乏咪唑。 </li><li>加咪唑至5mM的终浓度，以溶解的材料（步骤2.8），并且如果使用自动化液体色谱分析装置手动加载材料到柱上或到样品回路。 </li><li>加载溶解的材料在柱上以0.5ml /分钟的流速，并用2 CV咪唑缺乏缓冲洗在相同的流速，以除去未结合的物质。在50毫升猎鹰管收集馏分。 </li><li>在第一次洗涤中，使用3 CV的纯化缓冲液用40mM咪唑以1ml /分钟的流速。收集2 ml的组分。 </li><li>在第二次洗涤，用3 CV的纯化缓冲液用100mM咪唑。收集2 ml的组分。 </li><li>洗脱CFTR从3 CV的净化缓冲液400 mM咪唑的的HisTrap柱。收集2 ml的组分。 </li><li>在洗脱级分（步骤2.8）监测荧光。 </li><li>保留峰组分进行SDS-PAGE分析等分。闪光冻结剩余的峰值fraction样品并储存于-80℃，或继续下一个纯化步骤。 </li></ol> CFTR 5。凝胶渗透色谱（GPC）净化<ol><li>平衡色谱柱（6的Superose三百分之一十GL）与1.2 CV双蒸2 O其次是1.2 CV GPC缓冲区。 </li><li>在步骤5.1中，集中具有最高的GFP荧光使用100,000 MWCO离心式过滤器在4℃下在Ni-亲和纯化的馏分如果在净化DDM，避免集中0.3毫克/毫升的蛋白质的蛋白浓度高于样品，因为这会造成显著的样品损失。从浓缩器和离心机在100,000×除去滞留物 g离心30分钟，在4℃下沉淀的大颗粒。 </li><li>此示例注入到色谱柱和洗脱与1.2 CV GPC缓冲等梯度渐变。收集0.5毫升分数。 </li><li>测量GFP荧光在第2.8节，以确定那些含有CFTR的分数。保持小体积（ 如&lt;/ em&gt;的50微升）的每一个用于通过SDS-PAGE分析。 </li><li>冷冻在液氮中，并储存馏分在-80℃下</li></ol> CFTR的6。重构<ol><li>加脂质（步骤1.8），以​​纯化的CFTR在脂质与蛋白质之比为100:1（W / W）和孵育在4℃下1小时。类似地设置了脂质仅控制，具有进行GPC缓冲器相同体积代纯化蛋白质。 </li><li>使用疏水性吸附剂珠蛋白/脂质混合物去除洗涤剂。在5 CV双蒸2 O 5的CV 70％（V / V）乙醇，5 CV双蒸2 O和5 CV GPC缓冲器缺乏洗涤剂洗吸附剂珠。加200毫克每毫升的纯化蛋白的洗涤吸附剂珠孵育在4℃下过夜，轻轻摇动。 </li><li>从吸附剂珠重建样品收集到使用薄端的枪头一个新的管。 </li></ol> ATP酶活性的7。测量<ol><li>确定CFT率在96孔板形式使用改性Chifflet测定26,27 R-特异性ATP酶活性。用磷酸钠溶液（0.65毫摩尔）制备0-20纳摩尔磷酸盐在50微升的最终体积为标准。使用CFTR的缓冲区和ATP酶缓冲液1:1混合，以稀释磷酸股票。 </li><li>孵育两重建的CFTR和空白脂质体与1:100（体积/体积）ATP酶抑制剂（步骤1.9），冰浴10分钟。使用重构的CFTR中的至少5微克。 </li><li>添加ATP（步骤1.9）来为2mM的最终浓度和孵育在25℃下反应1小时。停止反应，加入40微升10％（重量/体积）SDS（步骤1.9），以每孔（包括标准）。 </li><li>加入100μl缓冲液A（步骤1.10），并孵育10分钟。加入100微升缓冲液B（1.10）至各孔，并测量吸光度为800nm​​，在96孔板兼容的紫外/可见分光光度计的波长。 </li><li>利用转换的吸光度在800nm​​处成游离的磷酸盐的量的磷酸盐标准。中减去背景信号（脂质体 - 仅孔）后计算ATP水解的速率。 </li><li>对于非重组CFTR使用CFTR的缓冲器的背景读数遵循相同的协议。 </li></ol>

<ol>
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作者没有竞争经济利益还是其他利益冲突就这项工作。

我们先前已经描述了鼠CFTR 14的过表达的方法。由于该协议的发布，我们已经表达和纯化CFTR几种不同直向同源使用相同的系统。所有测试的直向同源物迄今纯化以及在液化石油气洗涤剂，而DDM纯化表明在不同的直向同源物更多的变化（数据未显示）。这种灵活性说明了酵母的方法的力量：它可以筛选许多结构相对快速性，以选择一个适合某特定用途。 洗涤酵母微粒体用含有1M NaCl的前增溶与DDM结果在吸尘器微粒制剂，并减少污染物在稍后阶段的缓冲区。这一步是不必要的液化石油气协议作为最终的CFTR的样品纯度&gt; 90％无微粒冲洗。此外，净化DDM需要几个改动的缓冲区增溶一次提纯，即增加了额外的甘油和盐。一起，这些添加显着增加了DDM-溶解蛋白质的柱的结合。 DDM的纯化方法有改进的余地，尤其是去除那，判断通过质谱一个40kDa的主要污染物，是由于酵母核糖体亚基的L3，这似乎对镍树脂固有的亲和力。有在L3蛋白无明显polyHis序列，但检查其三维结构时结合到核糖体（PDB = 1FFK）示出了折叠的L3亚基具有潜在polyHis集群。这个乐队是在液化石油气纯化材料问题较少的原因可能是更严厉的液化石油气清洁剂。 虽然纯化DDM似乎是比在液化石油气较差，较温和的清洁剂如DDM可以是具有的功能和结构的分析更加兼容并且已经在几个透视晶的使用膜蛋白15-21的海洋学研究。此外，我们的研究结果显示，使用液化石油气导致了相对CFTR的ATP酶功能丧失，以净化DDM。因此，我们建议石油气为基础的纯化方案CFTR的产生，其中纯度是至关重要的，例如在应用，如翻译后修饰的表征，或在抗体的产生，液化石油气为基础的协议将被选择。在应用中的蛋白质的活性，并完全原生的状态是至关重要的另一方面，我们也提出了DDM的协议是一个更好的选择。 最后，该协议描述了CFTR中的两性离子洗涤剂石油气-14或非离子型洗涤剂DDM隔离一个可重复的方法。因此，它表示一个更大范围的纯化条件为CFTR比以前已报道10-13。此外毫克纯化的CFTR的数量可以是当具有高体积酵母生长系统，如20升发酵罐和一个大容量的细胞收集系统，如6升低速离心机转子组合使用这些方法获得。得到的CFTR具有裂解GFP标记，它允许在不同的生物化学和生物物理实验容易监测的蛋白质。 在这个手稿（含鸡CFTR的质粒或冻结的酵母细胞）中描述的试剂可以通过囊性纤维化基金会（美国）获得。

囊性纤维化（CF）是最常见的遗传性疾病在欧洲和北美约1 2,500活产婴儿的发病率。 CF时发生突变的囊性纤维化跨膜传导调节蛋白（CFTR），其功能在上皮细胞1的质膜蛋白引起的损失。这种缺陷最严重的后果是不可逆的肺损伤，缩短患者的预期寿命为&lt;40岁年龄2,3。 CFTR是一个ATP结合盒（ABC）转运，已演变成为一种离子通道1,4。尽管它在细胞的质膜相当的功能改变，但仍保留显著的序列同源性和其它的ABC转运。有趣的是，CFTR（ 即其监管区域和它的N-和C-末端）的专用零件不共享显著序列相似性与其他后生动物的ABC转运，因此没有线索，TH在CFTR这些序列电子商务的起源。其主要结构的基础上，CFTR被列为ABC转运蛋白家族的一个C-家庭成员，但对于剩余的功能联动，这个子系列没有强有力的证据。已经有谷胱甘肽转运活性的一些报道对CFTR 5-7，这将是与其他C-家族成员8,9的作用相一致，尽管其他报告表明，还原型谷胱甘肽可以抑制CFTR的ATP酶活性，而不是示出了底物诱导的刺激所特有的ABC转运10。离子电导的测量是足够灵敏，以允许单个CFTR分子的通道的活性进行研究1和CFTR通道属性已监测作为时间的函数，温度，ATP浓度，膜电位，磷酸化状态，以及在存在一系列的小分子抑制剂，增强剂和改性剂。这些研究也显著我们的ABC转运蛋白如何工作的知识补充。然而，CFTR的显著金额和其后续的纯化表达已被证明是特别具有挑战性和成功一直局限于几个实验室10-13。 开发更有效的药物，需要紧迫，但这个过程已经阻碍了缺乏CFTR纯化筛选小分子。解决CFTR的表达和纯化的问题将使高通量药物筛选旨在纠正在CF中的主要缺陷，也将打开的高分辨率结构研究路由信息来告知合理的药物设计。此外，蛋白质，例如其功能寡聚状态，相互作用蛋白和ATP酶活性的甚至相对基本的生物化学特性保持差的特点。之前我们已经报道了协议的绿色荧光蛋白和His标签的小鼠CFTR大型表达在S酵母 14，现在进一步描述了CFTR的净化方案。我们使用这些方法来净化5直向同源物CFTR的，并且本数据为鸡CFTR的纯化，例如，洗涤剂的溶解和纯化的选择是任意的膜蛋白的纯化的关键步骤。有几种洗涤剂筛选的CFTR中的溶解度，我们选择使用两种截然不同的洗涤剂中的纯化。十二烷基-β-D-麦芽糖苷（DDM）是一种非离子型洗涤剂已被广泛地用于膜蛋白15-21的结构和功能的研究。离子型洗涤剂1 -十四烷酰基-sn-甘油基-3 -磷酸-（1&#39;-rac-甘油）（LPG-14）是高效的CFTR的溶解，并在功能性膜蛋白10的纯化以前被使用过， 22,23，其中包括从S中提纯的CFTR 酵母24。&lt;/ P&gt;

<table border="0" cellpadding="0" cellspacing="0" style="width:492px;" width="491">
	<colgroup>
		<col />
		<col />
		<col />
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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:115px;">Company</td>
			<td style="width:161px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.2&#956;M syringe filter&#160;</td>
			<td style="width:115px;">Sartorius</td>
			<td style="width:161px;">FC121</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100 kDa MWCO centrifugal concentrator (PES membrane)</td>
			<td style="width:115px;">Vivaspin</td>
			<td style="width:161px;">VS0641</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2ml microfuge tubes</td>
			<td style="width:115px;">Sarstedt</td>
			<td style="width:161px;">72.695</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">40Ti rotor</td>
			<td style="width:115px;">Beckman Coulter</td>
			<td style="width:161px;"><a href="https://www.beckmancoulter.com/wsrportal/page/itemDetails?itemNumber=337901">337901</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50ml Sterile Falcon Tubes</td>
			<td style="width:115px;">Sarstedt</td>
			<td style="width:161px;">62.547.254</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:216px;">Adenosine triphosphate disodium salt (Na2ATP)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">A26209&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Liquid chromatography system</td>
			<td style="width:115px;">GE Healthcare</td>
			<td style="width:161px;">28-4062-64</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Aminoethylbenzenesulfonyl fluoride (AEBSF)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">A8456</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass bead-beating cell disrupter</td>
			<td style="width:115px;">BioSpec</td>
			<td style="width:161px;">1107900</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Benchtop centrifuge&#160;</td>
			<td style="width:115px;">HERMLE</td>
			<td style="width:161px;">Z300</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Benchtop centrifuge&#160;</td>
			<td style="width:115px;">Eppendorf</td>
			<td style="width:161px;">5417R</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Benchtop microfuge&#160;</td>
			<td style="width:115px;">Fisher</td>
			<td style="width:161px;">13-100-511</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Benzamidine hydrochloride</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">434760</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hydrophobic Beads SM-2 Adsorbent</td>
			<td style="width:115px;">BioRad</td>
			<td style="width:161px;">152-3920</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bromophenol blue</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">114391</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Centrifuge tubes</td>
			<td style="width:115px;">Beckman Coulter</td>
			<td style="width:161px;">357000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel imaging system</td>
			<td style="width:115px;">BioRad</td>
			<td style="width:161px;">170-808</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cholesterol</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">C8667</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chymostatin&#160;</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">C7268</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dimethylsulfoxide (DMSO)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">D8418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dithiothreitol (DTT)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">43815</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">E.coli total lipid extract</td>
			<td style="width:115px;">Avanti lipids</td>
			<td style="width:161px;">100500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Epoxysuccinyl-leucylamido-butane (E-64)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">E3132</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass beads, acid washed</td>
			<td style="width:115px;">Sigma</td>
			<td style="width:161px;">G8772</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol</td>
			<td style="width:115px;">Fisher</td>
			<td style="width:161px;">65017</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HisTrap HP columns (5 ml)</td>
			<td style="width:115px;">GE Healthcare</td>
			<td style="width:161px;">17-5247-05</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rapid Coomassie Stain</td>
			<td style="width:115px;">Novexin</td>
			<td style="width:161px;">ISB1L</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Centrifuge JA-17 rotor</td>
			<td style="width:115px;">Beckman Coulter</td>
			<td style="width:161px;">369691</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Leupeptin</td>
			<td style="width:115px;">Merck</td>
			<td style="width:161px;">108975</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Lyso-phosphatidyl glycerol-14 (LPG)</td>
			<td style="width:115px;">Avanti lipids</td>
			<td style="width:161px;">858120</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">MgSO4</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">M7506</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel tank SDS-PAGE system</td>
			<td style="width:115px;">BioRad</td>
			<td style="width:161px;">165-8006</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">n-dodecyl-&#946;-D-maltopyranoside (DDM)</td>
			<td style="width:115px;">Affymetrix</td>
			<td style="width:161px;">D310S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">S6191</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">NaN3</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">S2002</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">NH4Cl</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">A9434</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oligomycin</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">75351</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultracentrifuge</td>
			<td style="width:115px;">Beckman Coulter</td>
			<td style="width:161px;">392050</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Prestained protein standards</td>
			<td style="width:115px;">Fermentas</td>
			<td style="width:161px;">SM1811</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Desalting columns (Sephadex G-25)</td>
			<td style="width:115px;">GE Healthcare</td>
			<td style="width:161px;">17-0851-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pepstatin A</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">P4265</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phenylmethanesulfonylfluoride (PMSF)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">P7626</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sch28080</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">S4443</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium dodecyl sulfate (SDS)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">L37771</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium thiocyanate (NaSCN)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">251410&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel filtration 10/300 GL column</td>
			<td style="width:115px;">GE Healthcare</td>
			<td style="width:161px;">17-5172-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris-base</td>
			<td style="width:115px;">Formedium</td>
			<td style="width:161px;">TRIS01</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultracentrifuge tubes</td>
			<td style="width:115px;">Beckman Coulter</td>
			<td style="width:161px;">355618</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vortex mixer</td>
			<td style="width:115px;">Star Labs</td>
			<td style="width:161px;">N2400-0001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultrasonic water bath</td>
			<td style="width:115px;">Ultrawave</td>
			<td>F0002202</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Multimode plate reader</td>
			<td style="width:115px;">BioTek</td>
			<td style="width:161px;">BTH1MF</td>
		</tr>
	</tbody>
</table>

purification of the cystic fibrosis transmembrane conductance regulator protein expressed in saccharomyces cerevisiae

在囊性纤维化跨膜电导调节器（CFTR）蛋白缺陷导致囊性纤维化（CF），为常染色体隐性遗传病，目前限制了患者的平均寿命为&lt;40岁。新型药物分子的发展，恢复CFTR的活性在治疗CF的一个重要目标，而功能活跃CFTR的隔离是实现这一目标的一个有益的步骤。 我们描述了两种方法的CFTR的纯化来自真核异源表达系统，S.酵母 。像原核系统中，S。酵母可以迅速地生长在实验室以较低的成本，而且还交通和翻译后可以修改大量的膜蛋白。洗涤剂的溶解和纯化的选择是任意的膜蛋白的纯化的关键步骤。有几种洗涤剂筛选CFTR的溶解度，我们选择了两种合作ntrasting洗涤剂中的净化，使最终的CFTR准备进行调整以适应随后计划实验。 在该方法中，我们提供的CFTR的十二烷基-β-D-麦芽糖苷的纯化（DDM）和比较1 -十四烷酰基-sn-甘油基-3 -磷酸-（1&#39;-rac-甘油）（LPG-14）。蛋白质用这种方法在DDM纯化显示功能分析ATP酶的活性。蛋白质在LPG-14纯化，显示出高的纯度和收率，可以用来研究翻译后修饰，并且可用于结构的方法，如小角X-射线散射和电子显微镜。然而，它显示显著低ATP酶活性。

上述协议是为了隔离CFTR富集微粒体，与CFTR的几乎完全恢复了细胞破碎的粗微粒体和制备（ 图1）中的有效手段。其他细胞破碎方法也可以有效地使用。我们已经利用法式压力室，和其他high-pressure/cavitation设备（也与影响对目标的红宝石组合）具有同等的效率。为了方便和设备的初始成本低，我们找到了珠打浆方法是最好的。 使用液化石油气，以溶解和纯化，得到CFTR 80微克蛋白质/ L的培养在纯度&gt; 90％（ 图2）。高收益是由于CFTR由液化石油气（比较图2b，泳道2和4）高效溶解。此外，有效的和紧密结合到柱导致的CFTR中的未结合部分损失最小和缺乏CFTR在洗涤级分（ 图2，泳道3，图5和6）。洗脱的蛋白具有&gt; 90％的纯度，通过考马斯染色的SDS-PAGE凝胶估计，并使用CFTR和污染物带的光密度。凝胶渗透色谱（GPC）分离低分子量污染物，液化石油气和纯化的CFTR（ 图4，下图）。 该协议用 ​​于使用DDM CFTR的纯化给出了大约60％，大约50微克/升的产率（ 图3）的纯度。从GPC的负染的馏分洗脱，在约10ml（ 图4）的电子显微镜（EM）表明，DDM-CFTR的纯化包含20-30纳米直径的聚集体，以及10nm的直径（数据未示出）的较小的颗粒。这可能是小的聚集体可以可逆地相关联，并分解为超滤与1 MDA截止滤光器并没有消除对EM-检测的聚集体。液化石油气净化材料不吸附辉光放电电网，因此，研究了未染色的部分冷冻电镜。这表明一个相对小的尺寸的一个非常均匀的粒子群（6-8纳米的直径，数据未显示）。 最后，将纯化蛋白的ATP酶活性的测定（ 图5）。由于农行蛋白家族的一员，CFTR有两个核苷酸结合结构域（NBDS）能够结合和/或水解ATP。该数据表明，纯化的蛋白质是不能够水解ATP的液化石油气溶解的状态，并显示出弱的ATP酶活性的DDM的存在（ 图5中未填充的柱）。加入脂质和洗涤剂除去后，ATP酶的活性为4倍高对于已经在DDM（13纳摩尔ATP / min / mg蛋白）中纯化的样品。加入脂质和去除石油气同样还原活性的CFTR到已被使用液化石油气中分离，而是与一个最后的低速率（1.5纳摩尔ATP / min / mg蛋白）比DDM纯化和重组材料。 <img alt="图1"FO：内容宽度=“5英寸”FO：SRC =“/ files/ftp_upload/51447/51447fig1highres.jpg”SRC =“/ files/ftp_upload/51447/51447fig1.jpg”/&gt; 图1中的细胞裂解物鸡CFTR（CL）的监控级别，上清液（S）和在用于微粒体的分离和洗涤各种离心步骤芯块（P），SDS-PAGE凝胶上使用的凝胶荧光的绿色荧光蛋白的可视化标记。细胞破碎，离心在14000×g离心后取上清液包含几乎所有的CFTR（包括降解产物）。超速离心在200,000×g离心沉淀所有的全长CFTR留下一些片段在上清液中。超速离心在100,000 xg的盐洗微粒颗粒几乎所有具有去除一些进一步的片段CFTR。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51447/51447fig2highres.jpg" src="/files/ftp_upload/51447/51447fig2.jpg" /> 图2。纯化鸡CFTR在液化石油气通过固定化金属离子亲和层析的。的级分通过SDS-PAGE随后进行考马斯染色（上图）和荧光检测的GFP标记（下图）的分析。曲目：（1）微粒。 （2）液化石油气溶解微粒。 （3）未结合的物质。 （4）不溶性物质。 （5）及（6）40和100mM咪唑的洗涤。 （7）材料洗脱400 mM咪唑。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51447/51447fig3highres.jpg" src="/files/ftp_upload/51447/51447fig3.jpg" /> 图3纯化的鸡CFTR在DDM通过固定化金属离子亲和层析的馏分通过SDS-PAGE随后进行考马斯染色进行分析。左手面板之前洗脱显示分数。几个连续洗脱组分在显示在右侧面板与CFTR由箭头dicated。后来的级分中富集的40 kDa的污染物，已通过质谱法鉴定核糖体蛋白L3。 <img alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/51447/51447fig4highres.jpg" src="/files/ftp_upload/51447/51447fig4.jpg" /> 图4。通过凝胶渗透色谱纯化鸡CFTR的。CFTR由镍亲和层析纯化，浓缩，并应用到GPC柱。对CFTR（上图）的洗脱曲线纯化的缓冲液含有LPG-14（实线）或DDM（虚线）重叠。 SDS-PAGE（下图）显示，CFTR洗脱介于8和11毫升<img alt="图5" fo:content-width="5in" fo:src="/files/ftp_upload/51447/51447fig5highres.jpg" src="/files/ftp_upload/51447/51447fig5.jpg" /> 图5。ATP酶activit用改进的Chifflet检测26 ATP酶抑制剂的鸡尾酒的存在，以消除任何背景ATP酶活性的F-，P-和V型ATP酶（未填充的酒吧Ÿ纯化鸡CFTR的分数。蛋白DDM或液化石油气纯化的测定）。 ATP水解的速率洗涤剂去除和类脂添加（填充柱）后还测量。图中显示的平均值和标准偏差（n = 3）。平均值在存在和不存在脂质ATP酶活性，并在DDM和液化石油气业务的差异之间的差异是显著为p &lt;0.05。

Watch this Scientific Journal Video about Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae at JoVE.com

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

异源表达和囊性纤维化跨膜电导调节器（CFTR）的净化是显著的挑战和制约因素的药物治疗囊性纤维化的发展。这个协议描述了两种方法的CFTR的毫克量适合于功能和结构研究的分离。

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 ...

purification-cystic-fibrosis-transmembrane-conductance-regulator

Research

JoVE Journal

Biology

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

14.4K 次观看.  University of Manchester. 异源表达和囊性纤维化跨膜电导调节器（CFTR）的净化是显著的挑战和制约因素的药物治疗囊性纤维化的发展。这个协议描述了两种方法的CFTR的毫克量适合于功能和结构研究的分离。 

视频: Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed 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

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

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 ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

纯化 M。 magneticum应变AMB - 1磁小体相关蛋白MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

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 ...

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state.
The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor.
A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 &#181;M. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution. A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor.

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 ...