我们感谢为这项工作的资金通过其CFTR的三维结构联合会（授予数量FORD08XX0）的囊性纤维化基金会（CFF）。我们承认在这项工作的协会全体同仁的巨大贡献，特别是在设计的CFTR基因。我们也承认博士的宝贵贡献。詹姆斯·伯特利（NCSR Demokritos，希腊），马克·扬（英国卡迪夫大学，英国）和大卫·德鲁（伦敦帝国学院，英国），在工作的早期阶段。我们特别感谢手稿的关键读博士伊娜Urbatsch（德州理工大学，拉伯克）。

1。媒体和缓冲器的制备<ol><li>鼎贞：一升，暂停无氨基酸和0.77Ğ完整的补充混合物6.9克酵母氮基尿嘧啶在1升的水。高压灭菌。保存于4°C。 </li><li> YNBA：400毫升，暂停无氨基酸2.76克酵母氮基尿嘧啶和350毫升的水8克细菌琼脂0.38Ğ没有完整的补充混合。高压灭菌。 8克葡萄糖50毫升的水和热量，轻轻混合直到溶解。通过0.2微米的过滤器进行消毒，而琼脂熔化，添加到YNBA。在室温下储存。 </li><li> 20％葡萄糖液500毫升，500毫升鼎贞和热混合100克葡萄糖轻轻直到溶解。通过0.2μm过滤器传递到无菌的杜兰瓶。在室温下储存。 </li><li> 20％半乳糖培养基：2升，400克半乳糖轻轻混合2L鼎贞和热直至溶解。通过0.2微米的过滤器传递到无菌的杜兰瓶。储存在室温下的温升。 </li><li>蛋白酶抑制剂股：CFTR的是高度敏感的蛋白质4。笔者发现以下的抑制剂是有效地限制蛋白质，但读者不妨裁缝这个名单，以满足自己的要求。在100μL分装在-20°C储存，以减少冻融问题。所有抑制剂应稀释股票方案的工作浓度如下： </li></ol><table border="1"><tbody><tr><td> 抑制剂 </td><td> 股权集中度 </td><td> 备料 </td><td> 工作浓度 </td></tr><tr><td> AEBSF </td><td> 200毫米</td><td> 48mg溶于1毫升的蒸馏水</td><td> 0.2毫米</td></tr><tr><td>苯甲脒</td><td> 300毫米</td><td> 36mg溶于1毫升的蒸馏水</td><td> 0.3毫米</td></tr><tr><td>糜酶抑素</td><td> 4毫米</td><td>溶解在1毫升干马克的每公升2.5毫克苏</td><td> 4μM的</td></tr><tr><td> E - 64 </td><td> 7毫米</td><td>溶解在1ml蒸馏水的每公升2.5毫克</td><td> 7微米</td></tr><tr><td>亮肽素</td><td> 20毫米</td><td> 10毫克溶于1毫升的蒸馏水</td><td> 20μM的</td></tr><tr><td>胃蛋白酶抑制剂一</td><td> 15毫米</td><td>溶解在1毫升干二甲基亚砜10毫克</td><td> 15微米</td></tr><tr><td> PMSF </td><td> 1米</td><td>溶解在1毫升干二甲基亚砜174mg </td><td> 1毫米</td></tr></tbody></table><ol start="6"><li> DTT（1米）：154毫克二硫苏糖醇溶解在1ml蒸馏水。贮存于-20°C 1:1000稀释在缓冲区的使用说明。 </li><li> EDTA（0.5米）：100毫升的水混合29.22 g与乙二胺四乙酸。新增为10 N NaOH溶液滴加已经解散，直到所有的EDTA，pH值达到8。化妆水200毫升，并通过0.2微米过滤到无菌的杜兰瓶。在室温下储存。 </li><li>认证机构（300毫米的Tris-HCl，pH值7.4，0.56米的山梨糖醇，1毫米EDTA）溶解在350毫升的水18.17Ğ三基地，此外盐酸调整pH值至7.4。新增51.35Ğ山梨醇，使量高达500毫升的水。高压灭菌。加入1毫升EDTA原液在4和存储°C。 </li><li> CFTR的缓冲液（pH值8.0的50 mM Tris，10％V /至五甘油）：6.06ĞTris碱溶解于500毫升的水。用盐酸调节pH值至8，加入100毫升的甘油和弥补到1 L水。在4°C的高压灭菌器和存储</li><li> 2倍负载染料：50毫米的Tris-HCl（pH值7.6），5％甘油5毫米EDTA（pH值8.0），0.02％溴酚蓝，4％的SDS，0.05 M的数码地面电视。使10毫升，分装在-20°C和存储</li></ol> 2。筛选转化子该协议假定的CFTR-GFP-8His融合基因已插入到1到GAL1半乳糖启动（图），酵母质粒下游质粒已被改造成FGY217细胞，Pep4删除酿酒酵母突变体10 </em&gt;。细胞可以种植在几个星期YNBA板和储存在4°C对于长期储存，甘油股票应保存在-80°C。克隆和转化方法进行了详细介绍，德鲁等人 （2008）10。 <ol><li>从一个转换板挑5-10分隔殖民地。每个殖民地转移到一个单独的无菌50毫升猎鹰9鼎贞毫升和1毫升20％葡萄糖液管中。对于这一步，重要的是有一个文化（W / V），2％葡萄糖的最终浓度，以维持细胞的生长。一夜之间成长为16小时，在250转，30℃轨道摇床。 </li><li>使每个屏蔽殖民地的甘油库存。无菌条件下加入0.8毫升过夜培养至0.2毫升无菌甘油标记螺丝小瓶中，涡短暂存储在-80°C。 </li><li>稀释至最终体积为50毫升，余下的过夜培养中鼎贞，包括20％葡萄糖液250μL。对于这一步，重要的是要稀释血糖浓度约为0.1％（W / W）在文化，因为高糖能抑制GAL1启动10。标记的250毫升三角瓶中成长的文化困惑瓶0.7-0.8外径600 250转，30°C在轨道摇床。 </li><li>加入5毫升20％的半乳糖中每个烧瓶诱导的文化和生长16小时。 </li><li> CFTR的表达，用荧光显微镜确认。以100μL的文化，并添加100μL甘油溶液中的细胞迁移限制​​。在三角洲视觉RT恢复显微镜（或类似）分析细胞，用FITC标记过滤器（激发波长490 nm，发射波长528-538纳米）下的蓝色激光。应该在酵母细胞的细胞膜的荧光环的CFTR-GFP融合蛋白的阳性表达可见。未转基因酵母细胞可能被用来作为对照。 </li><li>转让文化进50毫升猎鹰管。细胞收获在3500 XG，4℃离心10分钟，在台式离心机。离心机运行的同时，准备2 ml离心管，用螺丝包含上冰约500μL酸洗玻璃珠和地方的上衣。弃去上清，每个颗粒和悬浮在500-800μL冰冷的CRB 同 蛋白酶抑制剂。转移到离心管，含珠悬浮并保持在冰上。 </li><li>裂解细胞，通过大力摇晃/涡旋每个离心管，搁在冰间期为30秒10期。一个beadbeater可以受聘作为一种替代，例如BioSpec迷你beadbeater经营3分钟。 </li><li>在3500 XG，4℃放置到台式离心，离心管5分钟。转移上清含粗膜人口microfuge管和地点清理冰。加入500μL新鲜冰合作LD认证机构 蛋白酶抑制剂，以每管和重复的过程中积累的膜。 </li><li>收集在2个小时的台式离心纺丝最大速度4°C，粗膜。弃上清，每个颗粒悬浮于50μL冰冷的CFTR缓冲区。 </li><li>在干净的离心管，15μL2X负载染料混合15μl每个悬挂的上下吹打起来。在室温下孵育10分钟。不开锅的样品，因为这将导致CFTR和其他膜蛋白形成SDS不溶性聚合和变性的GFP标记。 </li><li>另外到4-20％三/甘氨酸梯度凝胶电泳（NuSep）的PageRuler预染蛋白标准（Fermentas公司）一起装入样品和运行染料前，在150 V或40分钟，直到凝胶底部。 </li><li>凝胶荧光识别的最高表达。成如台风扫描仪荧光成像系统的地方。扫描凝胶ü唱激发波长在488纳米的蓝色激光的发射波长526纳米。 CFTR的GFP融合应该是在约220 kDa的可见光。也将是微弱的日光灯带，可见约70 kDa的，这可能是一种内在的酵母含有FAD的膜蛋白（如琥珀酸脱氢酶亚基）11,12。 </li><li>凝胶用考马斯染色，脱色和扫描凝胶进行比较，以荧光扫描，使用一个方便的图像浏览软件包。考马斯染色凝胶允许CFTR的绿色荧光蛋白表达水平在不同无性系正常化后的相对评估总蛋白量加载到每个凝胶的轨道。 </li><li>连续到一个新的YNBA了从甘油（2.2）的最高表达细胞株 板在30°C孵育2-3天。这个板块可能存储长达2周，在4°C </li></ol> 3。大型发酵罐邪教余吕杏茜<ol><li>准备在发酵前文化。刮从YNBA细胞1厘米2区 使用无菌环板（2.13）和添加45毫升鼎贞和5毫升20％葡萄糖液，这样的OD 600约为0.1。在250毫升锥形瓶莫名其妙增长轨道摇床，直到OD 600达到1 250转，30℃。 </li><li>分裂文化之间的两个2 L莫名其妙锥形瓶鼎贞每片含450毫升和25毫升20％葡萄糖液。种植这些在250转，30°C，直到在轨道摇床的OD 600达到1.2。 </li><li>这些预文化增长的同时，成立了发酵。如1.1中所述11.2升鼎贞，但解散额外Ğ鼎贞8.28和0.95Ğ下降了补充，以弥补此外甘油诱导。无菌增加11.2大号鼎贞和75毫升20％葡萄糖液无菌20L发酵容器和调整运行温度30°C。 </li><li>无菌的发酵的precultures添加和设置搅拌速度约800转，温度保持在30°C。压缩空气流入约15 DM 3分钟-1。一旦发酵培养达到1.2外径600，无菌条件下加入1.5升鼎贞20％半乳糖溶液和1.2升甘油诱导。降低温度至25°C和16小时成长的文化。 </li><li>转入1升使用蠕动泵的冰冷冻离心机盆发酵内容。收获细胞，在3500 XG，4℃离心30分钟，在一个大容量的转子（如6升）。悬浮细胞，在150毫升冰冷蛋白酶抑制剂的CRB。从这里开始，所有的工作应在4°C </li><li>常细胞破碎系统中的4张通行证，在25，30，32和35千帕，通过收集有关在每种情况下冰的裂解，裂解细胞。另外，使用珠·贝亚特·R与同等体积的0.5毫米酸洗玻璃珠（Biospec）和鼓动全功率为3分钟。离心裂解液转移至50ml猎鹰管和颗粒细胞碎片在3500 XG，4℃15分钟，在台式离心机。 </li><li>将上清转移至冷冻离心管。 14,000 XG，4℃离心30分钟，在离心机除去线粒体。 </li><li>将上清转移到冷藏的超高速离心管。在20万XG，4℃离心收集微粒的超高速离心90分钟。 </li><li>小心倒出，弃上清，并添加蛋白酶抑制剂和1mm数码地面电视2毫升冰冷的CFTR缓冲区，以每管。轻轻悬浮颗粒使用一个画笔，最高可达每管CFTR的缓冲区，并用旋涡混合器混合。 </li><li>在超高速离心60分钟的暂停为200,000 XG，4℃离心，丢弃上清，重悬浮颗粒在2毫升冰冷的CFTR的缓冲区瓦特第i个蛋白酶抑制剂 （没有DTT），使用画笔。 </li><li>池再悬浮微粒，调整最终体积至50毫升，CFTR的缓冲区，并拌匀。预订1毫升分装为SDS-PAGE电泳分析，如2.9中所述 - 2.11。微粒可以是闪光灯在液氮冷冻和保存在-80°C，直到所需要的。 </li><li> CFTR的可使用下列洗涤剂微粒，tetradecanoly，LYSO，磷脂酸锂perfluorooctonoate（LiPFO）（LPG14），N-十二烷基-β-D-麦芽糖苷（DDM）的提取。 CFTR的缓冲区，蛋白酶抑制剂和洗涤剂5％（W / W）的混合微粒。如果使用的DDM，也增加300毫米氯化钠的缓冲区。在4°C，搅拌15分钟，管肩。 </li><li>在超高速离心1小时，在10万XG，4℃离心样品。保留上清，并采取了SDS-PAGE电泳分析小等分。 CFTR可能现在被分离纯化固定金属从solublised材料的亲和层析其次是尺寸排阻色谱法。 </li></ol> 4。代表结果的质粒酵母与CFTR含有的转型是不是100％的效率。 CFTR表达的一个代表小规模屏幕中从转变实验选择的殖民地，将产生约1表达的蛋白质在4殖民地。从屏幕上从一个板块中挑选5殖民地的一个典型的结果显示在面板的图。 3。殖民地之一，显示了强烈的通常运行之间的250 kDa和130 kDa的标记，如下所示的CFTR-GFP融合蛋白表达水平。的CFTR GFP荧光水平将有很大的不同实验之间，图4殖民地。 3显示至少10倍更大的荧光比约70kDa的内在荧光带。如果CFTR的GFP表达水平出现给小于70 kDa的波段荧光，那么它可能是值得重新改造和选择一个殖民地CFTR基因-GFP的表达水平较高。正如图所示。 3A，这是不可能的，即使有CFTR的GFP表达水平高，是在考马斯染色的细胞中提取的CFTR-GFP波段可辨别。 一旦选定殖民地已种植规模较大的实验，以及微粒分离，CFTR的GFP在微粒的存在将需要进行评估，如图所示。 3B。这个实验的结果是重要的，不仅评估诱导表达的效率，而且还检查的微粒已精心准备和蛋白质已经被最小化。结果如图。 3B意味着，在这个实验中的CFTR-GFP的表达略低于在小规模的实验显示面板A.（如判断相对内在70kDa乐队），但这种印象是由荧光检测器在此过度偏颇测量。这是因为实验者是checki纳克的蛋白片段的CFTR构造的存在。有一些在这部分之间的130 kDa和100 kDa的标记荧光蛋白片段的实验证据，但这些都是非常薄弱相比全长CFTR的绿色荧光蛋白带。与蛋白酶抑制剂在这里，我们发现CFTR的蛋白降解，细胞破碎后，很少有证据。如果重要的蛋白质被发现，我们建议新鲜的蛋白酶抑制剂库存解决方案。我们还发现，商业蛋白酶抑制剂鸡尾酒片不为这个系统有效。细胞的生长超过16小时（诱导后）会引起CFTR表达下降，如在图4所示。这可能是由于营业额的蛋白质，可能是由于上调酵母蛋白质的质量控制机械6-9,13。因此，建议监察CFTR的表达水平与半乳糖诱导后，如果可能的话，作为最佳时间收获细胞的马Ÿ变化从一个实验室到另一个。 <img alt="图1" src="/files/ftp_upload/3860/3860fig1.jpg" /> 图1。 CFTR的结构含有酵母质粒 。 2μp424GAL1表达载体插入的CFTR-GFP-8His融合，半乳糖（GAL1）启动子的控制下。向量还包含一个的尿嘧啶选择标记（“市建局”）和氨苄青霉素抗性基因（安培）。 <img alt="图2" src="/files/ftp_upload/3860/3860fig2.jpg" /> 图2。流程图总结了可视化的协议。 <img alt="图3" src="/files/ftp_upload/3860/3860fig3.jpg" /> 图3。代表SDS-PAGE凝胶CFTR的表达和纯化。 A组显示五个随机挑选转化菌落（1-5车道），CFTR的表达筛选。B显示面板从15升FERM分离的微粒C组进入文化。显示两个阶段使用体积排阻色谱的亲和层析纯化后得到纯化的小鼠的CFTR。所有凝胶光照条件下，从GFP后，考马斯域（左）和激动人心的荧光染色（右）所示。分子量标准的相对位置都列在左边（KDA）。 <img alt="图4" src="/files/ftp_upload/3860/3860fig4.jpg" /> 图4。在酵母中表达的CFTR的有代表性的数据。 A组 CFTR的表达显示了一个半乳糖诱导后的时间，当然。细胞提取物经SDS-PAGE分析，集成的CFTR-GFP蛋白带GFP荧光。B组表达GFP的细胞16小时后诱导荧光显微镜显示了典型的结果。通常情况下，只有一小部分细胞表达高水平的CFTR。

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Cell. 18, 806-814 (2007).</li><li>Fu, L., Sztul, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ER-associated+complexes+(ERACs)+containing+aggregated+cystic+fibrosis+transmembrane+conductance+regulator+(CFTR)+are+degraded+by+autophagy.">ER-associated complexes (ERACs) containing aggregated cystic fibrosis transmembrane conductance regulator (CFTR) are degraded by autophagy.</a> Eur. J. Cell. Biol. 88, 215-226 (2009).</li><li>Sun, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derlin-1+promotes+the+efficient+degradation+of+the+cystic+fibrosis+transmembrane+conductance+regulator+(CFTR)+and+CFTR+folding+mutants.">Derlin-1 promotes the efficient degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) and CFTR folding mutants.</a> J. Biol. Chem. 281, 36856-36863 (2006).</li><li>Zhang, Y., Michaelis, S., Brodsky, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CFTR+expression+and+ER-associated+degradation+in+yeast.">CFTR expression and ER-associated degradation in yeast.</a> Methods Mol. Med. 70, 257-2565 (2002).</li><li>Drew, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFP-based+optimization+scheme+for+the+overexpression+and+purification+of+eukaryotic+membrane+proteins+in+Saccharomyces+cerevisiae.">GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae.</a> Nat. Protoc. 3, 784-798 (2008).</li><li>Knight, A. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis+transmembrane+conductance+regulator+degradation+depends+on.">Cystic fibrosis transmembrane conductance regulator degradation depends on.</a> the lectins Htm1p/EDEM and the Cdc48 protein complex in. 15, 4125-4135 (2004).</li><li>Yoo, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-conventional+trafficking+of+the+cystic+fibrosis+transmembrane+conductance+regulator+through+the+early+secretory+pathway.">Non-conventional trafficking of the cystic fibrosis transmembrane conductance regulator through the early secretory pathway.</a> J. Biol. Chem. 277, 11401-11409 (2002).</li><li>Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+the+cystic+fibrosis+transmembrane+conductance+regulator+protein+and+structural+changes+associated+with+phosphorylation+and+nucleotide+binding.">Architecture of the cystic fibrosis transmembrane conductance regulator protein and structural changes associated with phosphorylation and nucleotide binding.</a> Journal of Structural Biology. 167, 242-251 (2009).</li><li>Wellhauser, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small-molecule+modulator+interacts+directly+with+deltaPhe508-CFTR+to+modify+its+ATPase+activity+and+conformational+stability.">A small-molecule modulator interacts directly with deltaPhe508-CFTR to modify its ATPase activity and conformational stability.</a> Mol. Pharmacol. 75, 1430-148 (2009).</li><li>Ramjeesingh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+monomer+is+the+minimum+functional+unit+required+for+channel+and+ATPase+activity+of+the+cystic+fibrosis+transmembrane+conductance+regulator.">A monomer is the minimum functional unit required for channel and ATPase activity of the cystic fibrosis transmembrane conductance regulator.</a> Biochemistry. 40, 10700-10706 (2001).</li><li>Huang, P., Liu, Q., Scarborough, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysophosphatidylglycerol:+a+novel+effective+detergent+for+solubilizing+and+purifying+the+cystic+fibrosis+transmembrane+conductance+regulator.">Lysophosphatidylglycerol: a novel effective detergent for solubilizing and purifying the cystic fibrosis transmembrane conductance regulator.</a> Anal. Biochem. 259, 89-97 (1998).</li></ol>

没有利益冲突的声明。

本文提供了一个表达小鼠CFTR蛋白在酵母细胞中，它应该促进囊性纤维化研究的方法。这样做的目的是连接释放小鼠的CFTR基因的DNA结构，这将是通过囊性纤维化基金会（本文<a href="http://www.cff.org/research/CFFT/">http://www.cff.org/research/CFFT/</a> ）。其他同源应成​​为晚些时候推出。的与CFTR含载体的酵母细胞的转化是简单的，但重要的是屏幕CFTR的表达水平高的殖民地。可变的表达水平可能出现的几个因素，但质粒每个细胞的副本的数量可能占了显著的变化程度。这里所描述的关键步骤，应该允许CFTR的表达酵母细胞的CFTR含有微粒膜的生产。一旦转型，酵母细胞的生长，收获和裂解已掌握，purification的蛋白质应该是可能的，并在图3中，我们作为一个有用的基准，应该在这种情况下实现的纯度的一个例子。这不是我们在这个手稿的意向，提供详细的方法，蛋白质纯化。不过，也有一些关键的下游纯化步骤是特定的S.cerevisae表达系统，如细 ​​胞裂解和微粒净化，这些都被包括在这个手稿的详细。然而，应该提到的是，除了我们使用这两个方法，替代酵母细胞破碎方法可以聘用，如使用了法国的压力细胞。重组蛋白的裂解1 TEV-C-末端的绿色荧光蛋白域，使蛋白质被诱导后跟踪（图4）。酵母有一种内在的70kDa蛋白（有可能琥珀酸脱氢酶12）荧光在相同条件下11，这可以提供一个有用的跨CFTR的相对表达水平，在全细胞提取物或微粒的信号校准标准（图3）。它是由半乳糖诱导后，细胞收获时间是至关重要的，如图4所示的清晰数据。 CFTR的下降的产量急剧诱导后约16小时，以便有诱导24小时后检测的CFTR在酵母细胞中的任何勉强。 纯化蛋白的产量大约是1 - 2毫克每15升发酵罐培养的CFTR蛋白。可以恢复估计约70％的总CFTR的绿色荧光蛋白微粒阶段，纯化的蛋白质和25％左右恢复。表征的的S.酵母表达的CFTR是正在进行中。正如图。 4，蛋白质在细胞内的位置可通过荧光显微镜监测。虽然大部分的荧光预期10细胞的边缘周围发现，一些蛋白质会显示一个点状的本地化，无论是在或内质膜这可能是由于CFTR通过后期高尔基/内体通路14或者出芽从ER 4运输囊泡下游舱室回收。治疗与PNGaseF，的酶deglycosylates蛋白质，在SDS-PAGE电泳迁移的CFTR蛋白带的最小变化表明，这意味着它是unglycosylated，或具有最小的糖基化15。蛋白磷酸化状态的实验正在进行中。到目前为止测试的一些洗涤剂，纯化的蛋白显示ATP酶活性的（即由16的 CFTR特定抑制剂抑制）率是相似的原样2,15。的CFTR通道活性的测量，需要重组的纯化蛋白，这将意味着在最后一个洗涤剂的净化步骤，有相对较高的临界胶束浓度（CMC）17。酵母微粒containinĞ，CFTR可solublised几个普遍采用的洗涤剂18，包括清洁剂，如十二烷基麦芽糖2，被普遍认为是“温和”。然而，最高CMC洗涤剂已被证明是solubilsation低效，到目前为止，交流，到这些洗涤剂，应考虑在后期在任何净化计划。

<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录没有 </td></tr><tr><td> FGY217 酿酒酵母株，pep4删除10 </td><td></td><td></td></tr><tr><td>无氨基酸酵母氮基</td><td> formedium </td><td> CYN0410 </td></tr><tr><td>完成补充混合物没有尿嘧啶</td><td> formedium </td><td> DCS0169 </td></tr><tr><td>细菌培养基</td><td> Sigma-Aldrich公司</td><td> A5306 </td></tr><tr><td> D-半乳糖</td><td>费舍尔</td><td> BP656-500 </td></tr><tr><td> D-葡萄糖</td><td>费舍尔</td><td> D16-500 </td></tr><tr><td>胃蛋白酶抑制剂一</td><td> Sigma-Aldrich公司</td><td> P4265 </td></tr><tr><td>亮肽素</td><td>默克公司</td><td> 108975 </td></tr><tr><td>糜酶抑素</td><td> Sigma-Aldrich公司</td><td> C7268 </td></tr><tr><td> phenylmethanesulfonylfluoride（PMSF） </td><td> Sigma-Aldrich公司</td><td> P7626 </td></tr><tr><td> epoxysuccinyl leucylamido-丁烷（C-64） </td><td> Sigma-Aldrich公司</td><td> E3132 </td></tr><tr><td> aminoethylbenzenesulfonyl氟（AEBSF） </td><td> Sigma-Aldrich公司</td><td> A8456 </td></tr><tr><td>苯甲脒盐酸盐</td><td> Sigma-Aldrich公司</td><td> 434760 </td></tr><tr><td>二甲基亚砜（DMSO） </td><td> Sigma-Aldrich公司</td><td> D8418 </td></tr><tr><td>二硫苏糖醇（DTT） </td><td> Sigma-Aldrich公司</td><td> 43815 </td></tr><tr><td>乙二胺四乙酸（EDTA）的</td><td>费舍尔</td><td> BP120500 </td></tr><tr><td> Tris碱</td><td> formedium </td><td> TRIS01 </td></tr><tr><td>的Tris-HCl </td><td>费舍尔</td><td> P631 </td></TR&gt; <tr><td> D-山梨醇</td><td> Sigma-Aldrich公司</td><td> S1876 </td></tr><tr><td>甘油</td><td>费舍尔</td><td> 065017 </td></tr><tr><td>氯化钠</td><td> Sigma-Aldrich公司</td><td> S6191 </td></tr><tr><td> N-十二烷基β-D-maltopyranoside </td><td> Affymetrix公司</td><td> D310S </td></tr><tr><td>溴酚蓝</td><td> Sigma-Aldrich公司</td><td> 114391 </td></tr><tr><td> PageRuler加上预染蛋白标准</td><td> Fermentas公司</td><td> SM1811 </td></tr><tr><td> NuSep的Tris-甘氨酸4-20％梯度凝胶</td><td> NuSep </td><td> NB10-420 </td></tr><tr><td>即时蓝考马斯染色</td><td> novexin </td><td> ISB1L </td></tr><tr><td>玻璃珠，酸洗</td><td>西格玛</td><td> G8772 </td></tr><tr><td> 50毫升无菌猎鹰管</td><td>萨尔斯塔特</td><td> 62.547.254 </td></tr><tr><td> 2毫升无菌螺丝顶端小瓶</td><td>萨尔斯塔特</td><td> 72.694.005 </td></tr><tr><td> 250毫升无菌锥形莫名其妙瓶</td><td> BD公司</td><td> 355119 </td></tr><tr><td> 2升无菌锥形莫名其妙瓶</td><td> BD公司</td><td> 355131 </td></tr><tr><td> 2毫升离心管</td><td>萨尔斯塔特</td><td> 72.695 </td></tr><tr><td> 0.2μm的注射器过滤器</td><td>赛多利斯</td><td> FC121 </td></tr><tr><td>超高速离心管</td><td>贝克曼</td><td> 355618 </td></tr><tr><td>离心管</td><td>贝克曼</td><td> 357000 </td></tr><tr><td> 1升离心机盆</td><td>贝克曼</td><td> 969329 </td></tr><tr><td>轨道震动与温度控制的孵化器</td><td>新不伦瑞克省科技</td><td></td></tr><tr><td> deltavisionŕţ恢复显微镜</td><td>应用视觉</td><td></td></tr><tr><td>台式离心机</td><td> HERMLE </td><td> Z300的</td></tr><tr><td>台式离心</td><td>费舍尔</td><td> 13-100-511 </td></tr><tr><td>旋涡混合器</td><td> STAR实验室</td><td></td></tr><tr><td>台风三重奏扫描器</td><td> GE医疗集团</td><td> 63-0055-87 </td></tr><tr><td> LAS3000成像系统</td><td>富士</td><td></td></tr><tr><td> 20L发酵容器和控制单元</td><td> applikon </td><td></td></tr><tr><td>恒定系统细胞的破坏者</td><td>恒定系统</td><td></td></tr><tr><td> beadbeater细胞破碎</td><td> BioSpec </td><td> 1107900 </td></tr><tr><td>迷你beadbeater-16 </td><td> BioSpec </td><td> 607 </td></tr><tr><td> JA - 17罗托ŕ </td><td>贝克曼</td><td> 369691 </td></tr><tr><td>舰L - 100超速离心机</td><td>贝克曼</td><td> 392050 </td></tr><tr><td> 50.2Ti转子</td><td>贝克曼</td><td> <a href="https://www.beckmancoulter.com/wsrportal/page/itemDetails?itemNumber=337901" target="_blank">337901</a> </td></tr></tbody></table>

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

囊性纤维化跨膜电导调节器（CFTR）是氯离子通道，如果发生变异，可引起囊性纤维化humans.There是相当大的兴趣因此，在这种蛋白质，但研究其结构和活动的努力，已困难的阻碍表达和纯化足够数量的蛋白质1-3。像许多“困难”的真核生物膜蛋白，在一个快速增长的有机体表达是可取的，但具有挑战性的，在酵母学酵母 ，到目前为止，低量得到快速降解的重组蛋白，观察4-9。已在处理重组CFTR基因在酵母中的蛋白质参与6-9。在这份报告中，我们描述了一种方法在酵母和大量的净化CFTR的表达。协议描述如何可以克服早期的水解问题及如何表达水平通过修改细胞的生长条件，并通过控制诱导条件，CFTR可大大改善，特别是细胞收获前的一段时间。 reagants协议（CFTR的表达酵母细胞或酵母质粒）将通过美国囊性纤维化基金会，该基金会已赞助这一研究的分布式小鼠与此相关的。文章描述在本报告采用的设计和合成的CFTR结构将另行公布（Urbatsch，一;锡伯杜，体育等 ，未发表）。在这篇文章中，我们将解释我们的酵母细胞的CFTR建设与改造的方法开始 - 含有酵母质粒（图1）。该结构具有绿色荧光蛋白（GFP）序列融合在其C-末端CFTR如下德鲁等人开发的系统。（2008）10。允许CFTR的表达和纯化的GFP应遵循相对容易。朱庇特的可视化protocOL结束后，准备从酵母细胞中的微粒，虽然我们包括从微粒的蛋白质纯化的一些建议。读者不妨给自己加修改微粒净化过程，依赖于与蛋白质和向他们提供本地设备进行最后的实验。酵母表达的CFTR蛋白可以部分使用金属离子亲和层析纯化，用一种内在的多聚组氨酸纯化标签。随后体积排阻色谱产生一种蛋白质，它似乎是纯&gt; 90％，经SDS-PAGE和凝胶考马斯染色判断。

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

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

企图表达的囊性纤维化跨膜电导调节器（CFTR）<em&gt;酿酒酵母</em&gt;，到现在为止，已产生的蛋白质含量相对较低。通过囊性纤维化基金会分发本协议及相关的试剂应允许毫克这个&#39;困难&#39;真核生物膜蛋白的准备。

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  ...

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

18.4K 次观看.  University of Manchester. 企图表达的囊性纤维化跨膜电导调节器（CFTR）酿酒酵母，到现在为止，已产生的蛋白质含量相对较低。通过囊性纤维化基金会分发本协议及相关的试剂应允许毫克这个'困难'真核生物膜蛋白的准备。

视频: Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein 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 ...

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 ...

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 ...