Name: functional reconstitution and channel activity measurements of purified wildtype and mutant cftr protein
Uploaded: 2015-03-09T14:00:00.000+00:00
Duration: 9 min 59 s
Description: Watch this Scientific Journal Video about Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein at JoVE.com

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1.净化CFTR的注意:请参见表材料和设备的设备和材料在这个协议中使用的列表。存在重量的人，CFTR的过度表达和突变体在S F 9杆状病毒表达系统17,28的详细协议。过度CFTR并准备了S F 9细胞根据此协议的颗粒。 <ol><li>粗膜制备<ol><li>获取新的或从500毫升培养细胞过表达wildtype-，F508del-或G551D-CFTR蛋白的C末端His 10 -tag的解冻冷冻S F 9细胞沉淀，用标准的细胞培养方法进行培养，如前面描述的17,28。细胞小球将具有体积的约5 ml和大约5克湿重。 </li><li>重悬S F 9细胞在20ml磷酸盐缓冲盐水（PBS）pH 7.4的具有蛋白酶抑制剂混合物（1片每50毫升）中，在4℃下，确保了样品显示出目视均匀的并且没有团块的细胞的存在。 </li><li>使用高压细胞破碎（表材料和设备的），对于每通道2分钟达到最小10,000磅（优选15,000-20,000 psi）的裂解细胞。保持样品在4℃裂解过程中。 <ol><li>收集在裂解期间之后冰管中的样品，然后立即重新加载在细胞破碎样品。重复裂解过程3次。在显微镜下检查，以确保不超过10％保持不变。 注:其他的方法来裂解细胞，如玻璃珠等没有被严格地检查该系统中，然而，用户可能会发现这样的替代方法合适。 </li></ol></li><li>在4℃离心细胞裂解材料在高速离心机在2000×g离心20分钟。收集上清和处置的颗粒。划分上清液中20管和离心样品1小时，在4℃以125000×g离心以表格顶部ultracentrifuge。 </li><li>取出并弃去上清液，小心不要打扰颗粒，并在同一管店颗粒在-80℃下不到2个月，直至使用，或保留在冰上继续立即与净化。 </li></ol></li><li> CFTR溶<ol><li>获得1-4新鲜或冷冻S F 9粗膜颗粒和在1ml的缓冲液1的悬浮每个（2％FOS -choline; 见表1为完整配方）在4℃下在冰上，使用将25g针头和1毫升注射器，直到该溶液均匀肉眼不可见的细胞膜团块。其中，一个以上的小球被溶解，继续在平行于对待每说明书各样品为单个丸下面，汇集的样品在步骤1.3.2。 </li><li>溶解1小蛋白酶抑制剂片剂在1ml缓冲液2中（10mM咪唑; 见表1）它缺乏fos的 -choline 14洗涤剂（蛋白酶抑制剂是10倍以上concent额定比在这一点上他们推荐的稀释），并添加100微升的悬浮粗膜颗粒（蛋白酶抑制剂是他们推荐的稀释在这一点）。与由倒置混合5次，每次5分钟，置冰上45-60分钟。 </li><li>离心机以125000×g离心，4℃悬浮粗膜沉淀在台式超速离心机25分钟。保留上清液，其含有溶解的CFTR弃沉淀。 </li></ol></li><li>列绑定，磷酸化和洗脱<ol><li>洗400μl的镍NTA（次氮基三乙酸）琼脂糖浆或等效树脂用1毫升缓冲液2中（10 mM咪唑; 见表1）它缺乏洗涤剂，以除去溶剂和缓冲液，在4℃。重复洗两次。 </li><li>结合溶解的CFTR，其余的蛋白酶抑制剂（900微升）和镍树脂（400微升浆料）至总体积为10ml的缓冲液2（10mM咪唑; 见表1）瓦特HICH没有任何添加的洗涤剂。 （重量/体积）在此步骤中，FOS -choline 14浓度被有效地稀释到0.2％。如果有多个管，汇集所有样品含溶解的CFTR，蛋白酶抑制剂，镍NTA树脂和0.2％（重量/体积）FOS -choline-14在这一点上。孵育在4℃下60分钟，轻轻摇动。 </li><li>通过CFTR的蛋白酶抑制剂-镍NTA溶液向下一个小的筛选柱（ 表1）或等效的空柱。 </li><li>洗镍NTA树脂，其被保留在柱中，用缓冲液每3的初始沉淀4毫升（10mM咪唑+ DDM; 见表1）它缺乏fos的 -choline 14，但含有1mM的DDM（十二烷基麦芽糖苷清洁剂），在4℃下。此外DDM洗涤剂不显著改变这种缓冲液的pH。 </li><li>制备PKA-MgATP溶液中加入25微升（5毫米，最终浓度）MgATP的，2500的cAMP依赖性蛋白激酶A，催化亚基U（PKA），以475微升的缓冲液3（10mM咪唑+ DDM; 见表1）到柱上。通过密封用盖或封口膜柱的底端，并加入500微升PKA-MgATP溶液为每个使用的初始沉淀磷酸蛋白。 </li><li>在室温下孵育（20℃）下进行20分钟，温和混合，并使用1毫升吸移管，每2分钟的树脂的再悬浮，以确保PKA已经与镍NTA树脂的整个表面接触。 </li><li>取下盖和从柱上洗脱的PKA / MgATP溶液。用2ml缓冲液3中洗涤柱（10mM咪唑+ DDM; 见表1），在4℃。 </li><li>乘用在实验中通过4.洗涤所述柱，用毫升缓冲液4的这个数目颗粒的数目（50mM咪唑+ DDM; 见表1），在4℃，通过加入4毫升缓冲液的柱，使它流经并立即加入在接下来的4毫升一份到柱上。 </li><li&gt;洗脱的蛋白通过传递1毫升缓冲器5的（600 mM咪唑+ DDM; 见表1）以每通过该柱用1毫升的时间初始沉淀4℃没有暂停，以允许柱干燥，和收集含纯化，CFTR在单管整个洗脱液。 </li><li>浓缩蛋白样品用离心机过滤装置（100,000分子量截止），如在表材料和设备或其它类似设备的描述以去除咪唑和低分子量的降解产物，在总共为10ml缓冲液6（75毫KI + DDM; 见表1）或含有1mM DDM选择的其他缓冲液（如缓冲器7，用于非碘化流出试验中，25毫米的HEPES + DDM; 见表1），通过离心，在2000×g离心和4℃下进行约20分钟，直到样品浓缩至200微升。稀释样品至10ml并重复浓缩步骤。 注:根据我们的经验（未发表），咪唑显著INHI位的CFTR的ATP酶活性，并应在此步骤通过稀释和浓度的至少两倍去除如果样品将用于功能性的测量。 </li><li>收集集中净化CFTR。保持在4℃下在DDM缓冲液存在，直到使用在1-2天内或立即继续到重构步骤。不要冷冻纯化的蛋白质，在我们的经验，我们观察到减少活动。 </li></ol></li></ol> 2.重组CFTR的<ol><li>脂质准备 注:CFTR已成功重组到蛋的PC，POPC，2:1（重量/重量）鸡蛋PC:POPC（1:1 W / W）混合物，或PE的混合物:PS:PC:麦角甾醇，5:2 :2:1（W / W）。 <ol><li>对于碘化流出实验，干燥5毫克蛋的PC（200微升25毫克/毫升的库存，参见表材料和设备的）在氩气气流，在室温下20分钟，在一个派热克斯玻璃或其他坚固的（非硼硅）玻璃管中​​。 </li><li>使用280nm处的吸光度，将ELISA一个SSAY或其他方法，估计纯化的CFTR蛋白样品的浓度。对于碘化流出实验的野生型CFTR的，可以使用的蛋白质的1脂质比:300-1:3000（W / W），以产生一个足够的信号。对于G551D-和F508del-CFTR，使用蛋白质:200-1:约1脂质比1200（W / W），以检测流出活性。 </li><li>优化蛋白质:脂质的比例为每一个特定的系统。计算所需的纯化的蛋白的体积。计算缓冲器用1mM DDM体积，使1毫升， 即 1 ml的终体积所需- =缓冲液体积（所需蛋白质溶液体积）。 <ol><li>用1mM DDM添加所需的缓冲系统的计算量，以干燥后的脂质（但不添加此时CFTR蛋白）和盖用Parafilm玻璃管。对于碘化流出实验，重悬在缓冲液6脂质（75毫KI + DDM; 见表1）。对于其他的实验中悬浮缓冲区8英寸（25毫米HEPES + DDM，请参阅表1）或含有1mM DDM另一个所需的内部缓冲器。 </li><li>涡旋，在室温下2分钟，以帮助在悬浮液中的DDM缓冲器的脂质。或者，加热样品至37℃涡流前1-2分钟。 </li></ol></li><li>反复暂停所述脂质在室温下用1ml注射器和25g针，直到没有脂质膜是在管的两侧可见。避免将空气注入将产生细小气泡并有助于碘化物和脂质的氧化溶液中。 </li><li>超声处理在封口膜悬浮脂质覆盖管，用于在含水浴超声冰一个20秒的突发，然后立即转移到冰上1分钟。重复3次。 注:强烈的超声变成碘解决因氧化发黄。因此避免过度超声。监测样品的颜色变化。如果颜色变化指出，丢弃样品并再次准备。在颜色由于一些氧化所述变化碘化物将导致在相同条件下的脂质的氧化。取代从用氩气管中的空气进行超声处理的溶液中之前，将有助于防止脂质和碘的氧化。强烈超声处理可以打破质量差或导致样品损失划伤玻璃试管中。 </li><li>添加在步骤2.1.3计算到超声处理脂质样品到一个纯化的CFTR的量达到1毫升的最终体积。通过再悬浮混合与脂质和洗涤剂溶液中的蛋白质的10倍与地下25针和1ml注射器。 </li><li>在冰上30分钟，以管的旋流设置脂类和蛋白质样品混合5次，每次5分钟。 </li></ol></li><li>洗涤剂结合柱的准备<ol><li>获得在单次使用商业洗涤剂结合旋转柱（见表材料和设备的）与1毫升体积容量并打破底部标签以启动柱流出。 </li><li>离心2分钟列在2000×g离心以除去存储器Buffer，并丢弃该缓冲区。 </li><li>加入5 ml缓冲液7（75mM的KI， 见表1）的柱中，然后离心200×g离心4分钟，以洗脱所述洗涤缓冲液。重复此步骤2次以上，总共3次洗涤，以除去所述存储缓冲器的所有痕迹。丢弃所有流量通过。 注意:用于洗涤塔的缓冲区不包含DDM或任何其它的洗涤剂，这一点至关重要。列具有用于洗涤剂有限结合能力并且如果使用含有洗涤剂的缓冲液，该列将是无效的，在从蛋白质 - 类脂的洗涤剂样品中除去洗涤剂。 </li><li>仔细分装所有将1ml脂质洗涤剂蛋白样品上柱树脂的顶部和孵育样品在塔的顶部，在室温下2分钟。 </li><li>离心样品，在室温下搅拌4分钟，在2000×g离心，并收集在一个干净的1.5或2 ml离心管混浊脂蛋白体样品或玻璃管，并放置在SAMPLE冰上。 </li><li>通过加入200μl的缓冲液7（75mM的KI， 表1）或其他缓冲液（无去污剂）与塔的顶部收集在塔其余脂蛋白体，并重复离心步骤，持续2分钟。 </li><li>添加第二洗脱液的脂蛋白体样品，如果它看起来浑浊。 </li><li>存储所述样品在4℃下过夜或立即用于碘化物流出测量。 </li></ol></li></ol> 3.碘外排的测量纯化，冲调CFTR <ol><li>整个proteoliposomal膜碘梯度代<ol><li>预溶胀的Sephadex G50珠粒在缓冲液9（75mM的K-谷氨酸;谷氨酸钾， 见表1），（在50ml缓冲液约5克干燥的珠子），或所需的外部缓冲在室温下至少2小时或在4 °过夜。 </li><li>准备在缓冲液9的饱和4的Sephadex G50柱（75毫米的K-谷氨酸， 见表1）或其他缓冲区小筛查列加载树脂至少¾全在列。 </li><li>离心4分钟，在2000×g离心来从树脂去除多余的缓冲区。应该有大约2.25毫升在所述离心步骤的所述端部的柱填充水合树脂。 注:树脂应该轻易的水分，但不能浸泡在液体和随后的简短旋不应洗脱显著体积的缓冲液中。这是对脂蛋白体样本列树脂既不是太湿也不能太干，用户可根据需要进行实验的列，以熟悉最成功的缓冲区交换条件成功洗脱关键。 </li><li>小心在2000×g下125-150微升脂蛋白体的样品添加到每个柱和离心机的顶部为4分钟。 </li><li>游泳池脂蛋白体洗脱液一起在碘外排或其他实验立即使用。 注意:从每个柱洗脱的体积应大致150微升和样品应该有点浑浊，但比原来的样品少雪。较大体积或明确洗脱液表明列没有被充分干燥，或已被干燥的太多，分别和不会导致一个成功的碘化物流出实验。 </li></ol></li><li>碘外排法<ol><li>洗碘化物选择性微电极（表材料和设备的）广泛地与去离子水，然后在实验缓冲液10之前孵育20分钟（15μMKI， 见表1）。用去离子水再广泛冲洗电极。 </li><li>添加150微升的药物（20μM的典型浓度以产生10μM的在测定中的最终浓度）在缓冲液9（75mM的K-谷氨酸， 见表1）的96孔板的或车辆在缓冲液9到一个阱与小跳蚤搅拌棒。 <ol><li>确保没有泡沫存在。用枪头消除杂散气泡。如果正在使用的药液，保证在阱中的最终DMSO浓度为小于1％（体积/体积）（通常0.1-0.2％（体积/体积））。 </li></ol></li><li>添加150微升被制作在部分3.1的缓冲液9重构CFTR蛋白（75毫K -谷氨酸， 见表1）至很好地与药物或缓冲液中，以产生在所述板的孔中加入300微升的总体积。 </li><li>放置在96孔板上的搅拌盘和搅拌在130转（或近似最大速度的四分之一在中速）。 </li><li>插入碘化物选择性电极的尖端仔细地进入井与样品使得该尖端不接触孔的底部或被击中的搅拌棒。确保参考电极，如果分开，也与在孔中的溶液接触。 </li><li>使用与电极接口自制或商业计算机程序的记录轨迹（见表材料和设备的详细信息）。通过低通滤波器使用2 Hz的采样率，与信号的滤波。 </li><li>允许该样品平衡5分钟，以产生一个稳定的平面，或接近平面的基线录制时。 </li><li>加入3微升MgATP（100毫制备的dh 2 O和调节至pH 7，用KOH的库存; 1mM的终浓度）与样品以激活蛋白。让〜5分钟，以达到一个新的稳定的基线。随时调整的100mM MgATP股票的pH至中性后再使用，以避免改变在外部缓冲液的pH。 </li><li>加入3微升缬氨霉素存量（2μM; 20nM的终浓度）与样品分流的变化，通过CFTR由碘化物选择性电导产生的电势差。记录的下降斜率（减少以mV）由于CFTR介导的碘化流出活性为至少5分钟。 </li><li>以确保碘化物在脂蛋白体腔的捕集充足，加10％的3微升（体积/体积）的Triton X-100的样品。 SignificanT和即时碘的释放表示有充足的碘已经被困在这样诱捕不是在3.2.9确定的变化斜率而是CFTR介导活动的限制因素的囊泡。 </li><li>治疗用药物或用Triton X-100的样品后，用去离子水彻底清洗电极和搅拌棒，以确保这些试剂的所有痕迹已被删除，以避免下一个样品的污染。 </li><li>制备一系列稀释的碘化钾浓度以微摩尔至纳摩尔范围的缓冲液9（K -谷氨酸缓冲液， 见表1）。记录电极的每个浓度的毫伏响应从0到制备的最高浓度。建立标准曲线，其中所述探测响应（毫伏）绘制对lo​​g摩尔碘化物浓度的。使用标准曲线来转换流出实验碘化物浓度释放期间探头的毫伏响应。 注:准备校准CUR已经每周直到用户是肯定的速度有多快的探测器的变化的响应。会有在探针随时间的响应和灵敏度漂移。作为探针的年龄，响应会降低。这可通过周期性地改变内部溶液和探针尖端的水充分洗涤后使用，这有可能限制分子与探针表面的粘附部分地废止。 </li><li>确定的浓度对时间曲线图用于加成MgATP的之前的最后30秒每脂蛋白体样品的直线的平均斜率，和加法缬氨霉素后但加成的Triton X-100的之前。减去基线斜率从缬氨霉素，以确定为该样本的CFTR介导的碘化物流出活性。 </li></ol></li></ol>

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作者承认这里所述的纯化方法，开发过程中Mohabir Ramjeesingh医生的意见。这些研究是由运营赠款从健康研究（CIHR）和囊性纤维化加拿大（CFC）的加拿大学院CEB支持。 PDWE是通过在CIHR和CFC奖学金奖励部分支持。作者承认由囊性纤维化基金会治疗（CFFT）提供校正，增强剂和R·布里奇斯博士（医学罗莎琳德大学版）抑制剂化合物和分布。作者还通过贝勒细胞培养基金（NIH资助P30 CA125123）使用杆状病毒表达系统的重量和突变CFTR蛋白承认出色的服务。无效VX-770模拟，V-09-1188，是Vertex制药公司的礼物。

已经出现了纯化流程为全长，功能性CFTR隔离数量有限，从各种细胞过表达系统。此处所描述的方法是有利的，因为它允许将Wt-CFTR或F508del-和G551D-CFTR的中等量的高富集的快速纯化即高功能的测定中，包括ATP酶和通道功能的直接测量，包括在平面双层单信道测量系统和人口CFTR通道功能表现的措施是高度相关的小分子19-21的研究。纯化方法被集成到一个快速有效的重建协议，从其他的方法但是...

跨越上皮细胞在这样的组织如肺，肠，胰腺和汗腺的顶膜的氯离子转运主要由囊性纤维化跨膜传导调节因子（CFTR），一个ATP-和磷酸化调节的部件的基础知识（A​​TP-介导的结合膜蛋白的录像带）C亚科（1回顾）。像ABCC亚科的其他成​​员，CFTR是一个大的，多生成树整合膜蛋白，其在形成在其核苷酸结合结构域（NBDS），在那里它具有适度的ATP酶活性，在一个单一的接口两个核苷酸结合位点结合的ATP站点。然而，与其他ABCC亚家族成员，CFTR已演变为一种独特的监管氯通道而不是作为一个活跃的溶质转运。 CFTR突变导致囊性纤维化，这种疾病影响多个器官包括肺，胃肠道，胰腺和生殖道，导致morbidity和死亡率在青壮年。肺部疾病通常占在囊性纤维化早期死亡率和在大多数情况下是由CFTR功能上的传导气道表面上皮的损失。 CFTR氯离子通道活性的缺乏导致在两个氯的减少-横跨表面上皮和水的运动来修改流体层上的纤毛呼吸上皮的顶端表面。这导致粘稠的气道表面液体，可以削弱纤毛呼吸道上皮细胞的能力，以有效地清除病原体从气道。其结果是，大部分的CF患者患有肺部感染和肺损伤引起炎症的反复发作。 正如预期的那样，正常的CFTR蛋白的作用机制研究主要集中在其通道门控活动的详细的电生理研究。单信道研究表明直接说CFTR用作PKA依赖性Cl-它拥有一个ATP调节的2号门通道。详细的电生理学研究提供了大量的关于单个CFTR通道1,3的信息，但是可能有关心是否已被研究的任何特定单个信道的特性是反射性CFTR通道的全部人口，因此单信道的结果应始终与方法来研究宏观的人口考虑沿。纯化CFTR的人口通道活性的直接检测有可能提供洞察与致病突变相关的分子缺陷，并带动化工发现其调制器修复突变CFTR蛋白。迄今为止，有超过在CFTR 1900不同的突变被认为造成囊性纤维化4。主要的突变，F508del-CFTR，发现至少一个等位基因的患者，在北美和欧洲的约90％会导致蛋白质错误折叠一第二保留于内质网5。 F508del-CFTR也有其他后果，包括有缺陷的通道活性6-9。从细胞表面所得到的不存在的CFTR的与严重的疾病有关。 G551D-CFTR，一个不太常见的突变，被认为是正确折叠但是不正常如在细胞表面6一氯离子通道。小分子校正和增效剂的发展具有校正的折叠和/或贩卖的突变体，如F508del-CFTR到细胞表面的，和增效或增加突变的通道活性如G551D在细胞表面上存在时，目标分别。而校正的VX-809和VX-661（尚未批准使用的患者，所述增效剂Kalydeco（ivacaftor; VX-770）正在使用，在150毫克每12小时在CF患者&gt; 6年与至少一个G551D -CFTR突变，以及最近的患者G178R，S549N，S549R，G551S，G1244E，S1251N，S1255P之一和G1349D。 Kalydeco是既安全又导致改善的疾病的CF 10的临床措施，这种小分子的作用却是知之甚少在FDA批准用于在患者使用时的机制。 少数CFTR纯化方法先前已被描述2,11-18，其中许多需要相当长的时间才能完成。在最近的出版物19，一个独特的快速纯化和重组方法被用于过度表达的CFTR中的S F 9细胞表达系统描述的，并且在所定义的脂质系统本纯化的蛋白被用来建立一个CFTR卤化物通道活性测定为CFTR的人口分子。该测定概括的磷酸，核苷酸和抑制剂对CFTR功能的已知作用。该系统被用来询问增强剂的效果的VX-770 / Kalydeco上重量（野生型），F508del-和G551D-CFTR和它表明用于第一时间，该药物直接交互的CFTR蛋白，以使可能在一个ATP无关的方式其信道活动，从总体角度来证明这些方法的实用性和适用性CFTR与核苷酸和小分子的突变体的相互作用的研究回答关于蛋白质临床相关的问题。该方法也被用来研究其它增强剂分子和它们的衍生物20中 ，以及对蛋白质21的活性的小分子校正的效果。 流出测定法已被用于在许多研究中预先调查的CFTR突变体的活性和CFTR-调节化合物在其活性的影响，包括使用的电极，放射性示踪剂和荧光团22,23的全细胞测定法，膜囊，用离子选择性电极24和纯化重组CFTR与放射性示踪剂25。然而THE使用的离子选择性电极，研究纯化重组CFTR首次最近19日报道。当前方法的改编已用于重建和绿脓杆菌 ，一个共同的CF病原体2的膜蛋白的功能特性。加之碘化流出测量纯化ALGE外膜蛋白的重组被用来通过此传送机构26，以支持一个模型阴离子藻分泌。重构和碘化流出测量施加到纯化WZX蛋白，其允许被提出了一个模型，通过该蛋白27表明一个H +依赖性反向转运机制为跨细菌内膜的脂质连接寡糖的易位。在这两种情况下，碘化用作替代物的阴离子基底，尽管在较低的吞吐量比人们所预料要天然底物。该方法可以适用于适应其它蛋白质与Cationic运输或传导途径阴离子基底。 这里一个快速纯化过程被描述为CFTR蛋白和其重组到所定义的脂质脂蛋白体。迅速重建过程可以很容易地定制用于与CFTR通过其他方法纯化的使用，其前提是在纯化中使用的洗涤剂的种类是适合于除去由这里所使用的方法，也可以用于重建过程之前合适的去污剂交换。碘化物流出方法纯化和重组CFTR蛋白的通道活性的测定进行详细说明，并可以从该方法得到的一些典型结果。

<table><tbody><tr><td>&lt;em&gt;fos&lt;/em&gt;-choline 14 detergent</td><td>Anatrace Affymetrix (www.anatrace.affymetrix.com)</td><td>F312</td><td>Affymetrix: Anatrace Products; CAS#&amp;nbsp;:77733-28-9</td></tr><tr><td>cOmplete EDTA-free protease inhibitor cocktail tablets</td><td>Roche (www.roche-applied-science.com)</td><td>04 693 132 001</td><td>EDTA-free Protease inhibitor cocktail tablets (1 in 50 ml or mini: 1 in 10 ml) (Roche Diagnostics GmbH; Ref: 11873 580 001)</td></tr><tr><td>cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpack&amp;nbsp;</td><td>Roche (www.roche-applied-science.com)</td><td>05 892 791 001</td><td></td></tr><tr><td>Ni-NTA Agarose</td><td>Qaigen GmbH</td><td>1018240</td><td></td></tr><tr><td>Fisherbrand Screening columns</td><td>Fisher Healthcare</td><td>11-387-50</td><td></td></tr><tr><td>Amicon Ultra Centrifugal filters, Ultracel-100K</td><td>Millipore (www.millipore.com)</td><td>UFC910008</td><td></td></tr><tr><td>cAMP-Dependent Protein Kinase A (PKA), Catalytic Subunit</td><td>New England Biolabs (www.neb.com/products/p6000-camp-dependent-protein-kinase-pka-catalytic-subunit)</td><td></td><td>Peirce PKA is also suitable</td></tr><tr><td>PC, Chicken Egg</td><td>Avanti Polar Lipids (www.avantilipids.com)</td><td>840051C</td><td></td></tr><tr><td>POPC</td><td>Avanti Polar Lipids (www.avantilipids.com)</td><td>850457C</td><td></td></tr><tr><td>PS, Porcine Brain</td><td>Avanti Polar Lipids (www.avantilipids.com)</td><td>840032C</td><td>CFTR has been successfully reconstituted into Egg PC, POPC, 2:1 (w/w) Egg PC:POPC, or a mixture of PE:PS:PC:ergosterol, 5:2:2:1 (w/w)</td></tr><tr><td>PE, Chicken Egg</td><td>Avanti Polar Lipids (www.avantilipids.com)</td><td>841118C</td><td></td></tr><tr><td>Ergosterol</td><td>Sigma (www.sigmaaldrich.com)</td><td>45480</td><td></td></tr><tr><td>Pierce Detergent removal spin column</td><td>Thermo Scientific</td><td>87779</td><td>1 ml capacity columns</td></tr><tr><td>valinomycin</td><td>Sigma (www.sigmaaldrich.com)</td><td>V-0627</td><td></td></tr><tr><td>VX-770 (ivacaftor)</td><td>Selleck Chemicals</td><td>S1144</td><td></td></tr><tr><td>iodide selective microelectrode</td><td>Lazar Research Laboratories (www.shelfscientific.com/cgi-bin/tame/newlaz/microionn.tam)</td><td>LIS-146ICM</td><td>&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Clampex 8.1 software</td><td>Axon Instruments (www.axon.com)</td><td></td><td>we use components of the ClampX system with a home made filter to monitor and record the response to our electrode</td></tr><tr><td>Alternate Software: ArrowLabb System&amp;nbsp;</td><td>Lazar Research Laboratories (www.shelfscientific.com/cgi-bin/tame/newlaz/ionsystems.tam)</td><td>LIS-146LICM-XS</td><td>Lazar Research sells a meter that can interface with a computer and software to record the probe response.&amp;nbsp; This software should serve a similar function to our setup.</td></tr><tr><td>small stir bars</td><td>Big Science Inc (www.stirbars.com)</td><td>SBM-0502-CMB</td><td>choose a stir bar small enough to easily fit into a well of a 96-well plate</td></tr><tr><td>Sephadex G50, fine</td><td>GE Health Care (www.gelifesciences.com)</td><td>17-0042-01</td><td></td></tr><tr><td>Sonicator</td><td>Laboratory Supplies Co, Inc.</td><td>G112SP1G</td><td>Bath sonicators from other manufacturers should also be suitable</td></tr></tbody></table>

functional reconstitution and channel activity measurements of purified wildtype and mutant cftr protein

囊性纤维化跨膜传导调节因子（CFTR）是ATP的一个独特的通道形成构件结合转运盒（ABC）家族。 CFTR的磷酸化和核苷酸依赖的氯离子通道活性已经常研究整个细胞系统和单频道切除膜补丁。许多囊性纤维化致病突变已显示出改变该活动。而少数的纯化方案已经发表，即保留通道活性的快速重建的方法和一种合适的方法来研究人口通道活性的纯化的系统已缺乏。这里迅速的方法描述用于纯化和全长CFTR蛋白的功能重建成保留活性调节的卤化物信道定义的脂质组合物的蛋白脂质体。这个重构方法与通道活性的新颖的磁通为基础的检测一起是一个合适的系统，用于研究的野生型CFTR的人口信道特性和致病突变F508del-和G551D-CFTR。具体地，该方法在研究磷酸，核苷酸和小分子如上CFTR通道活性增强剂和抑制剂的直接影响实用。该方法也适合于其他的膜通道/转运对阴离子基底的研究。

描述这个写入出版物中一些方法来纯化，重建并测量CFTR蛋白的调节的通道活性。 图1A示出的工作流程的纯化，重组和碘化流出程序。方法用于重建和通道活性的测量通过碘化通量也列于进一步的细节，在相关联的视频。 净化CFTR和重建成脂蛋白 CFTR可以功能性以高水平表达于S F使用含有WT或突变体的CFTR基因2重组杆状病毒9细胞。...

Watch this Scientific Journal Video about Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein at JoVE.com

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

这里描述的是一种快速，有效的方法为纯化的野生型和突变型CFTR蛋白的功能性重组，可以保留活动而此氯离子通道，这是有缺陷的囊性纤维化。从由CFTR介导的重组蛋白脂质体碘流出允许通道活性的研究和小分子的影响。

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

functional-reconstitution-channel-activity-measurements-purified

Research

JoVE Journal

Biology

12.1K 次观看.  Hospital for Sick Children. 这里描述的是一种快速，有效的方法为纯化的野生型和突变型CFTR蛋白的功能性重组，可以保留活动而此氯离子通道，这是有缺陷的囊性纤维化。从由CFTR介导的重组蛋白脂质体碘流出允许通道活性的研究和小分子的影响。 

视频: Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

光谱分析 TRPV1 的纯化与重构

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

科研

生物化学

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

实时测量核苷酸与完整功能性膜蛋白的结合

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

生物学

<meta charset="utf8"></head><body>基于 FRET 的无电生理学监测 LRRC8 通道活性的方法

Monitoring Leucine-Rich Repeat Containing 8 Channel (LRRC8/VRAC) Activity Using Sensitized-Emission F&#246;rster Resonance Energy Transfer (SE-FRET)

Members of the LRRC8 protein family form heteromeric ion and osmolyte channels with roles in numerous physiological processes. As volume-regulated anion channels (VRACs)/volume-sensitive outwardly rectifying channels (VSORs), they are activated upon osmotic cell swelling and mediate the extrusion of chloride and organic osmolytes, leading to the efflux of water and hence cell shrinkage. Beyond their role in osmotic volume regulation, VRACs have been implicated in cellular processes such as differentiation, migration, and apoptosis. Through their effect on membrane potential and their transport of various signaling molecules, leucine-rich repeat containing 8 (LRRC8) channels play roles in neuron-glia communication, insulin secretion, and immune response. The activation mechanism has remained elusive. LRRC8 channels, like other ion channels, are typically studied using electrophysiological methods. Here, we describe a method to detect LRRC8 channel activation by measuring intra-complex sensitized-emission F&#246;rster resonance energy transfer (SE-FRET) between fluorescent proteins fused to the C-terminal leucine-rich repeat domains of LRRC8 subunits. This method offers the possibility to study channel activation in situ without exchange of the cytosolic environment and during processes such as cell differentiation and apoptosis.

<meta charset="utf8"></head><body>使用敏化发射 Förster 共振能量转移 （SE-FRET） 监测含有 8 通道亮氨酸的富含亮氨酸重复序列 （LRRC8/VRAC） 活性

Electrophysiology is the gold standard for investigating ion channel activity. However, there are plenty of alternative approaches, including optical methods. Here, we describe a method to monitor the activity of the leucine-rich repeat containing 8 channel (LRRC8)-formed anion channels using an inter-subunit F&#246;rster resonance energy transfer (FRET)-based method.

使用敏化发射 Förster 共振能量转移 （SE-FRET） 监测含有 8 通道亮氨酸的富含亮氨酸重复序列 （LRRC8/VRAC） 活性

Members of the LRRC8 protein family form heteromeric ion and osmolyte channels with roles in numerous physiological processes. As volume-regulated anion ...

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  ...

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4 and secretory diarrhea5. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut7.
Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)20. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18.
Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29.

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

依赖的PDZ-CFTR的大分子信号复合物的体外分析

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

重组脂膜的结构和功能研究的钾离子通道

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

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;

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

摘要

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此视频中的章节

光谱分析 TRPV1 的纯化与重构

实时测量核苷酸与完整功能性膜蛋白的结合

异源表达在哺乳动物细胞离子通道的突变和功能分析

使用敏化发射 Förster 共振能量转移（SE-FRET）监测含有 8 通道亮氨酸的富含亮氨酸重复序列（LRRC8/VRAC）活性

使用敏化发射 Förster 共振能量转移（SE-FRET）监测含有 8 通道亮氨酸的富含亮氨酸重复序列（LRRC8/VRAC）活性

使用敏化发射 Förster 共振能量转移（SE-FRET）监测含有 8 通道亮氨酸的富含亮氨酸重复序列（LRRC8/VRAC）活性

囊性纤维化跨膜传导调节蛋白表达和纯化酿酒酵母

依赖的PDZ-CFTR的大分子信号复合物的体外分析

重组脂膜的结构和功能研究的钾离子通道

囊性纤维化跨膜电导调节蛋白表达纯化

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

摘要

探索更多视频

此视频中的章节

光谱分析 TRPV1 的纯化与重构

实时测量核苷酸与完整功能性膜蛋白的结合

异源表达在哺乳动物细胞离子通道的突变和功能分析

使用敏化发射 Förster 共振能量转移 （SE-FRET） 监测含有 8 通道亮氨酸的富含亮氨酸重复序列 （LRRC8/VRAC） 活性

使用敏化发射 Förster 共振能量转移 （SE-FRET） 监测含有 8 通道亮氨酸的富含亮氨酸重复序列 （LRRC8/VRAC） 活性

使用敏化发射 Förster 共振能量转移 （SE-FRET） 监测含有 8 通道亮氨酸的富含亮氨酸重复序列 （LRRC8/VRAC） 活性

囊性纤维化跨膜传导调节蛋白表达和纯化酿酒酵母

依赖的PDZ-CFTR的大分子信号复合物的体外分析

重组脂膜的结构和功能研究的钾离子通道

囊性纤维化跨膜电导调节蛋白表达纯化

使用敏化发射 Förster 共振能量转移（SE-FRET）监测含有 8 通道亮氨酸的富含亮氨酸重复序列（LRRC8/VRAC）活性

使用敏化发射 Förster 共振能量转移（SE-FRET）监测含有 8 通道亮氨酸的富含亮氨酸重复序列（LRRC8/VRAC）活性

使用敏化发射 Förster 共振能量转移（SE-FRET）监测含有 8 通道亮氨酸的富含亮氨酸重复序列（LRRC8/VRAC）活性