这项工作是部分来自印第安纳大学 - 普渡大学印第安纳波利斯研究扶持资金补助（RSFG），部分由美国心脏协会14GRNT20390154奖，支持ARK

1.细菌表达。 注意:在此研究中所用的表达质粒在表1中描述。在这项研究中使用的解决方案，媒体，和缓冲液在表2中描述。古菌蛋白酶体亚基基因的克隆和表达质粒的产生在其他地方18,20描述。总之，对于亚基共表达重组质粒采用双顺反子操纵子的策略，这有助于获得各个亚基18,20可比表达水平。下面列出的表达式参数为凭经验确定为是最佳的本研究中的蛋白酶体亚基。可能有必要，以优化表达为其它蛋白酶的突变体，对蛋白酶体从其他古细菌物种，或者用于其它重组蛋白质复合物（见讨论）。 <ol><li>变换的利益表达质粒导入化学感受大肠杆菌 BL21（DE3）细胞。 </li><li>加入1 - 质粒2微升（一般为50 - 100纳克）到DE3细胞的等分该被新鲜在冰上解冻，并继续在冰上孵育20分钟。 </li><li>热休克的细胞在42℃下45秒，并返回管到冰上additionl 2分钟。 </li><li>加入1毫升的LB培养基中，并在37℃用1小时摇动（150rpm）下孵育。 </li><li>价差100 - 200μL转化混合物的到LB-kan平板（含固体LB培养基，辅以卡那霉素片（在本研究中编码的电阻用于这种抗生素所有质粒））。在37ºCO / N板。 </li><li>第二天早上，接种从LB-Kan平板的单一菌落到3mL的液体LB根介质中的玻璃培养管中。在37ºC孵育约2.5摇动（150转） - 3小时，或观察到混浊。 </li><li>测量在分光光度计600纳米（OD 600）的培养物的光密度。稀释细胞崇拜茜与预热的LB根介质的适量的6ml的终体积的0.4的OD 600。恢复在37ºC振摇40分钟。 </li><li>通过从原液加入IPTG到1mM的终浓度诱导在液体培养物的蛋白质表达。在37ºC孵育约6振荡（150转） - 7小时。 </li><li>在收获的全部细菌细胞培养到1.5毫升离心管。 </li><li>添加1.5毫升培养成离心管中。离心机以10,000×g的1分钟。 </li><li>弃上清，另一个1.5mL的相同培养物添加到粒料。离心机以10,000×g的1分钟。 </li><li>重复步骤1.11，直到整个文化收集到离心管。 </li><li>在-80℃，直到裂解存储诱导粒料。 </li></ol> 2.细菌裂解和裂解液混合。 注意: </strong&gt;的对于通过共表达研究的样本，请按照2.1节（及其子段）。对于需要裂解液混合样品，请按照2.2节（及其子段）。被包括在该协议中的TSP（共，可溶性，球团）的分析是优化蛋白质表达有用，它可以帮助确保α和β亚基裂解物混合（见讨论）期间被组合的大约相等量。它还提供了一个重要的控制，如果下游的结果并不如预期（ 即它验证蛋白表达和可溶性）。因此，我们强烈建议包括TSP分析，初步。一旦最佳协议已被用于特定亚基组合实现，TSP分析没有严格要求这就是为什么它被描述为以下可选的。 <ol><li>细菌细胞和制备可溶性部分的裂解。 <ol><li>在冰上解冻的诱导细胞沉淀5分钟。重悬在裂解缓冲液的600微升沉淀。 </li><li>公司乌瓦特在30°C以上的悬浮液振荡（150rpm）下进行30分钟，以产生总的粗裂解物。 注意:下面的步骤和随后的小节是可选的。 </li><li>从总的原油裂解成单独的1.5毫升离心管中取出两台25微升等分并进行分析TSP如下。 <ol><li>在两个25微升等分试样中的一个中，添加5X SDS样品缓冲液为1X的终浓度。标签管带"T"的"总原油裂解液"。 </li><li>孵育100ºC"T"型样品5分钟以完全变性蛋白质。 </li><li>同时，在第二个25微升等份，并在10,000×G下离心它10分钟。小心取出上清液，而不会干扰细小颗粒，并将其转移到一个新的1.5 ml离心管。标签"可溶部分"这一新管"S"。 </li><li>要细小颗粒离开behi次在前面的工序中，添加裂解缓冲液和旋涡的25μL到重悬。标签此管"P"为"颗粒部分"。 </li><li>添加6 5XμL的SDS样品缓冲液以"S"和"P"的管，并如上所述在100ºC孵育。 注意:如果TSP样本将不被使用的那一天来分析表达，它们可在-20 C，直到需要冷冻。一旦解冻，它们必须在100ºC被重新培养，如上所述，立即加载之前在12％和/或15％的标准SDS-PAGE凝胶。 </li></ol></li><li> ，同时TSP分析进行时，离心机以10,000×g的10分钟（如果TSP分析不进行或总粗裂解物的整个600微升）剩余550微升总粗制裂解物。收集上清在一个新的1.5 ml离心管。这是将被用于纯化可溶性裂解物。 </li></ol></li><li>裂解液混合。 <ol><li>在步骤2.1.1和2.1.2以上描述进行裂解。 </li><li>从表达来自表达所需β亚基细菌总粗裂解物的600微升的所需α亚基细菌混合总粗裂解物的600微升。在37ºC孵育缓慢振荡30分钟。 注意:可能需要优化的温育时间，实现裂解物混合期间最大组件（见讨论）。的时间，此处提供温度是最佳的用于本研究的重组蛋白并18别处测定。 </li><li>离心机以10,000×g的10分钟的混合溶解物可溶的材料从不溶的沉淀分离。将上清转移到新的1.5毫升离心管中。使用蛋白质纯化这种混合溶于裂解液。 </li></ol></li></ol> 3.通过固定化钴亲和树脂蛋白纯化（ICAR）。 <ol><li>平衡的树脂。 <ol><li>通过反转瓶彻底重悬树脂并传输的浆液的50微升到1.5mL离心管中。离心机在700×g下2分钟以沉淀树脂。小心吸上清。 注意:我们开展使用蓝1毫升枪头除去大部分液体的移液器吸出。白2尖μL用于去除剩余的上清液的精细控制，留下一个非常薄的覆盖珠液层。 </li><li>加1缓冲液A混合的溶液轻轻地在700×g下悬浮树脂和离心2分钟以沉淀树脂。小心吸上清，重复此清洗步骤一次。 </li></ol></li><li>适用于部分2.1或2.2的平衡树脂先前获得的可溶性裂解液。孵育轻轻旋转管中于4ºC60分钟。离心管中，在700×g下&lt;/ em&gt;的5分钟，然后小心地吸出上清液。 </li><li>洗涤树脂如下除去非特异性结合的蛋白质。 <ol><li>用1mL的缓冲液A和悬浮树脂孵育在4ºC温和摇动10分钟。离心在700×g下离心管5分钟，并小心地吸出上清液。重复该洗涤步骤一次。 </li><li>用1毫升缓冲液B（缓冲液A 5 mM咪唑）悬浮树脂并在4ºC轻轻摇动5分钟孵育。离心在700×g下离心管5分钟，并小心地吸出上清液。重复该洗涤步骤一次。 </li><li>用1毫升缓冲液C（缓冲带10毫米咪唑）悬浮树脂并在4ºC轻轻摇动5分钟孵育。离心在700×g下离心管5分钟，并小心地吸出上清液。 </li></ol></li><li>加入400缓冲液E的μL（缓冲液A与200毫米咪唑）洗脱蛋白质Ť澳树脂。在4ºC轻轻摇动5分钟孵育。离心机在700×g下5分钟。 </li><li>传输含有纯化蛋白上清至新的1.5毫升离心管中。 </li><li>通过串行离心脱盐纯化的蛋白质如下。 <ol><li> 400μL纯化蛋白，加入100微升缓冲液A（这减少了从200毫米到160毫米咪唑浓度）。应用纯化的蛋白至0.5毫升超离心过滤器，用10 kDa的分子量截止。离心机在14000×g下5分钟。 </li><li>弃滤液，加入400μL缓冲液A再次稀释截留和离心机。继续离心/稀释的循环，直至咪唑浓度低于4毫米。作为一个例子，如果每个离心浓缩500微升至〜70微升（或约7倍），那么两个周期将减少开始160 mM咪唑浓度（7×7 = 49倍）到大概oximately 3.3毫米。 </li><li>测量使用BCA测定法21脱盐样品的蛋白质浓度。 注意:脱盐需要减少到容限以下为BCA测定咪唑水平，如在制造商的说明。我们的实验室更喜欢BCA测定，因为它是敏感，有一个大的动态范围，并且表现出少得多的蛋白质与蛋白质的变化。但是，其它的方法来确定蛋白质浓度可以取代的BCA测定。关键是要了解每种方法的优点和局限性。 </li></ol></li><li>添加5X天然样品缓冲液为1X的终浓度，然后继续电泳。另外，店内样品在-20ºC供以后分析。 </li></ol> 4.非变性聚丙烯酰胺凝胶电泳（PAGE）。 注意:未聚合丙烯酰胺是一种神经毒素。穿戴适当的保护GEA河<ol><li>准备非变性PAGE胶如下。 <ol><li>准备凝胶盒使用清洁玻璃板铸造，铸造立场。放置在一个小磁力搅拌器之上梯度壶和放置一个微小的搅拌棒到每个室。 </li><li>用40％（重量/体积）丙烯酰胺和2％（重量/体积）二丙烯酰胺储备液，制备5％和10％（重量/体积）在天然解决缓冲丙烯酰胺凝胶溶液。确保总丙烯酰胺到双丙烯酰胺之比为37.5:1。冰浴前浇筑。 注意:这里使用的非变性PAGE系统对拉姆力SDS-PAGE系统基本相同，与SDS省略22。 </li><li> （（从10％（重量）在水中制备/ v）的原液）中添加过硫酸铵的丙烯酰胺溶液以引发聚合。过硫酸铵的最终浓度为0.1％（重量/体积）。 </li><li>倾丙烯酰胺溶液进入梯度制造者的两个腔室。随着出口管插入between凝胶盒的板，激活磁力搅拌器和打开梯度制造者阀。倒入5 - 10％非变性梯度凝胶。 </li><li>浇一次，覆盖所述凝胶与异丙醇的薄层，并允许凝胶聚合30分钟。在此期间，准备在当地解决缓冲新鲜的5％丙烯酰胺凝胶的解决方案。凝胶集后，倒出异丙醇和从喷射瓶冲洗用去离子水凝胶的顶部。 </li><li>向上述制备的5％的凝胶溶液中，添加过硫酸铵（恰好在4.1.3中描述），并在聚合的凝胶的顶部倾直到玻璃板是满的。将凝胶梳子。允许重叠凝胶聚合另外30分钟。 </li><li>一旦凝胶聚合，组装胶盒插入电泳仪。可替代地，存储在4ºC凝胶，包裹在湿纸上的毛巾和保鲜膜，直到准备使用。 </li></ol></li><li>一旦非变性PAGE凝胶上被组装到电子角trophoresis设备，填补1X本地运行缓冲罐准备从10X本地运行缓冲库存的新鲜。 </li><li>纯化的蛋白质的负载10微克，在段3的端部获得的，向每孔用玻璃注射器。 </li><li>负载2微升的高分子量天然蛋白质标准（在1X天然运行缓冲液稀释）插入孔中的一个。 </li><li>在55 V和4ºC运行凝胶，直到染料前沿跑出凝胶（约4 - 4.5小时）。 </li><li>除了非变性凝胶，分析由标准的SDS-PAGE 22纯化蛋白质的等分试样。 <ol><li>混合纯化的蛋白质样品，在段3的端部获得的，具有5X的SDS样品缓冲液的等分试样（10微克）至1X的终浓度。 </li><li>孵育100ºC5分钟，并加载到标准的12％，和/或15％SDS-PAGE凝胶22。 </li><li>在80V持续20分钟，运行，然后在120伏，直到染料前沿跑出凝胶（这大约是一个附加itional 75分钟为12％的凝胶，和120分钟为15％的凝胶）。 </li></ol></li></ol> 5.可视化活性和蛋白质染色。 <ol><li>电泳后，仔细分离的玻璃板和非变性凝胶转移到含有50毫升的去离子水的凝胶托盘。冲洗凝胶轻轻摇动5分钟。倒掉水，重复此清洗步骤三次。 </li><li>如下执行基板覆盖法。 <ol><li>添加1毫升显影含有荧光肽底物SUC-LLVY-AMC缓冲液中，均匀地分布在凝胶中。在37ºC30分钟。 注意:玻璃棒或凝胶释放器（用来分离玻璃板塑料小楔）可用于在凝胶分散液。 </li><li>小心地将凝胶转印到凝胶成像系统的紫外透射并观察荧光由于裂解的肽底物。记录图像。小心地转移凝胶巴放至凝胶托盘。 </li><li>用50毫升去离子水5分钟温和摇动冲洗凝胶的两倍。 </li><li>染色用10mL胶体考马斯染色试剂的凝胶60分钟温和摇动。用50毫升水凝胶脱色直到背景变得清晰。此染色步骤适用于标准SDS-PAGE凝胶，以及。 </li></ol></li></ol>

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The authors have nothing to disclose.

我们展示相结合的办法的好处通过重组古蛋白酶非变性PAGE分析蛋白酶体装配。蛋白酶体亚基的细菌共表达的常用方法9,11允许快速分析，但可能不露出键组件的中间体。我们建议用裂解液共表达相结合的混合开发汇编事件更宽阔的视野。 这种结合方法的优点是，尽管要求的α分开的表达和β亚基为裂解物混合，它仍然是相对劳动友好。结果也可以是半定量如果确保了单个蛋白质的可比较的和一致的表达水平。为此，我们建议进行重组蛋白质表达的通常的优化来确定允许之前裂解物混合表达的蛋白质的可比较的水平的条件。优化可以包括不同的感应恬即，诱导温度，诱导时，细菌表达菌株的光密度，并依此类推，直到可溶性蛋白的所需水平得以实现。进行比较的蛋白的野生型和突变的版本时，因为有时突变可以表现出可比的表达水平，但降低的溶解度，这是特别重要的。我们也强调确定ICAR纯化样品的蛋白浓度的重要性加载非变性PAGE之前。即使在地方表达的优化，并采取确保平行样品通过以相同的方式纯化步骤处理最好的照顾，仍可以在不经意间引入方差。的浓度测量确保了总蛋白的相同量的每个样品装载孔。这使得带强度的车道到车道的比较对于给定的迁移物种更有意义。 如果表达的可比性和一致性水平不能lysA的实现TE混合，人们总是可以净化所有组件事先并开展纯蛋白质混合实验。这具有允许更准确的蛋白质测定的优点，从而使该方法的定量。然而，充分纯化的缺点是，过程变得相当多的劳动强度大，它可以排除多个突变同时18的快速分析。值得一提的最后一个需要注意的是，有时分离α的表达和β亚基可能是不可能的。如果一个突变导致亚基自身表达时成为不溶性的在大肠杆菌中 ，但允许当所述突变体与它的结合配偶表示要恢复的溶解度可能出现这种情况。如果发生这种情况，它会限制分析只共表达。然而，这是可以在一般的重组蛋白质表达过程中产生一个警告，并且不是特定于古细菌蛋白酶体亚基24,25。 我们选择了根中心提供全方位我们应有的缓解ICAR树脂纯化和承受能力的蛋白酶体的蛋白他标记的衍生物。其它表位标签是可能的，包括那些基于抗体的纯化，我们已经成功地表达，我们的蛋白酶体亚基的纯化Flag标记的版本（未示出）。然而，如果需要纯化同质性（或增加生产规模），在他的标记版本提供了这样做的最快和最具成本效益的手段。 它是一个给定的，与重组蛋白获得的结果，无论是古细菌或在细菌中产生真核蛋白，将进一步获得当与体内观察随访意思。然而， 在体内的方法可能不总是实验立即访问。这是特别真实时研究的主题是一个大的，多亚基复合物如蛋白酶。重组蛋白方法为浮图的重要启动点再实验。在蛋白酶体的情况下，配对重组古蛋白酶生产与非变性PAGE将继续阐明蛋白酶体组装，这是古和真核生物9,11,12,18之间共享的关键特征非常有效。这样的方法可能是用于研究等大型多蛋白复合物的装配以及有用。最近，我们使用这种策略证明古细菌蛋白酶体可以通过一个以上的通路装配，以及α-环是未组装期间专性中间体，它们被认为是18。它仍然如果同样适用于真核蛋白酶体真正确定。

多蛋白复合开展众多关键的细胞活动1。对于许多这些配合物，进一步被知道关于其结构和功能大于约他们的组件2,3。蛋白酶体就是这样的一个复杂的，在生活的各个领域中找到。在真核生物中，这种分子机是在泛素/蛋白酶体系统（UPS）的核心，并提供了细胞内蛋白质的降解4的主要途径。真核蛋白酶体（简称为26S蛋白酶体）包括两个主要的子组件:一个20S蛋白酶，或核颗粒（CP）的5，可以在通过一个19S调节颗粒（RP）6的一端或两端封端。 20S蛋白酶是一个大的条块蛋白酶。其四级结构跨越所有生活领域绝对保守和由含有两种类型的结构上相关的亚基四七元环的栈，α;和β5,7,8。在真核生物中，这两个外圈各自包括的七个不同的α亚基和两个内环各自包括的七个不同的β亚基;蛋白水解活性驻留三个β亚基的范围内。相比之下，古细菌和细菌的CP环通常仅包括一种类型的α和一种类型的β亚基组成。古菌蛋白酶提供了一个重要的模型系统来研究蛋白酶体装配，由于双方的成分简单，他们与真核同行9-13共享一个共同的组装机理。简言之，α亚基组装成α环第一，它作为其上β亚基组装的支架。所得半蛋白酶（α7β7）二聚化，从而产生完全组装的CP（α7β7β7α7）。在二聚化，前肽提出关于β亚基被自催化除去，露出催化N-末端苏氨酸。利用古蛋白酶体模型组装经常需要生产中大肠杆菌重组古蛋白酶蛋白质的优势。这是一个值得的方法，因为它使在不同的组合要生产的亚单位，因为WT和突变的版本中，在不产生其自身的蛋白酶的宿主生物体。 监测多蛋白质复合物的装配生化需要某种用于分隔组件的中间体和前体的完全组装络合物分馏方法。由于其优越的解决容量，非变性聚丙烯酰胺凝胶电泳（PAGE）已被证明是在各种大型多蛋白复合物14-17的分馏特别有用。重组古蛋白酶生产和非变性PAGE的结合已成为一个dissectin强大的方法摹蛋白酶装配9,11,12,18。然而，通过此方法应用于（ 即通过α和β亚基的重组共表达）通常的方法有一个重要的缺点。大会的反应是合作，并强烈浓度依赖性3。鉴于在细胞内的蛋白质浓度是非常高的19中 ，由于排除体积效果，装配反应在体内迅速进行。因此，它有可能错过重要组件中间体当α和β亚基同时表达。 在这里，我们主张用重组古蛋白酶体亚基组装蛋白酶的研究组合的方法。在这种方法中，采用两个共表达和溶胞产物混合方法。前者允许组装的快速分析，因为共同表达是低劳动强度。后者取决于α和β亚基，接着混合的分开的表达。虽然这requi水库比共表达多一点努力，其大于补偿用于通过检测被共表达时错过中间体的能力。总之，这两个方法可以提供蛋白酶组件的更全面的了解。

<table><tbody><tr><td>Acrylamide (40%) solution</td><td>Biorad</td><td>1610104</td><td>Unpolymerized acrylamide is a neurotoxin. Wear proper protective gear</td></tr><tr><td>Amicon ultra 0.5 mL centrifugal filters</td><td>EMDMillipore</td><td>UFC501024</td><td></td></tr><tr><td>Ammonium Persulfate</td><td>Sigma</td><td>A3678</td><td></td></tr><tr><td>ATP</td><td>Sigma</td><td>A7699</td><td></td></tr><tr><td>BCA assay kit</td><td>Pierce</td><td>23225</td><td></td></tr><tr><td>Bisacrylamide (2%) solution</td><td>Biorad</td><td>1610142</td><td></td></tr><tr><td>Bromophenol blue</td><td>Sigma</td><td>B8026</td><td></td></tr><tr><td>DNaseI</td><td>Sigma</td><td>DN25</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Thermo Fisher</td><td>BP172</td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; BL21 competent cells</td><td>EMD Millipore</td><td>69450</td><td></td></tr><tr><td>GelCode Blue</td><td>Thermo Fisher</td><td>24592</td><td>Colloidal coomassie stain reagent for gels</td></tr><tr><td>Gel doc EZ system</td><td>Biorad</td><td>1708270</td><td>Gel documentation system</td></tr><tr><td>Gel releasers</td><td>Biorad</td><td>1653320</td><td>Wedge shaped plastic used to separate gel plates; useful for spreading liquid.</td></tr><tr><td>Glass rod</td><td>Thermo Fisher</td><td>11-380B</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>49767</td><td></td></tr><tr><td>Glycine</td><td>Thermo Fisher</td><td>BP3865</td><td></td></tr><tr><td>Hamilton syringe</td><td>Thermo Fisher</td><td>14-813-38</td><td>Glass syringe for loading gels</td></tr><tr><td>HEPES</td><td>US Biologicals</td><td>H2010</td><td></td></tr><tr><td>HMW Native calibration kit</td><td>GE Healthcare</td><td>170445-01</td><td>High molecular weight protein standards</td></tr><tr><td>Hoefer SG30</td><td>Thermo Fisher</td><td>03-500-277</td><td>Gradient maker</td></tr><tr><td>Imidazole</td><td>US Biologicals</td><td>280671</td><td></td></tr><tr><td>IPTG</td><td>US Biologicals</td><td>I8500</td><td>For induction of protein expression</td></tr><tr><td>Isopropanol</td><td>Thermo Fisher</td><td>BP26181</td><td></td></tr><tr><td>Kanamycin sulfate</td><td>US Biologicals</td><td>K0010</td><td></td></tr><tr><td>Lysozyme</td><td>Sigma</td><td>L6876</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fluka analytical</td><td>630680</td><td></td></tr><tr><td>Mini Protean Tetra Cell</td><td>Biorad</td><td>1658002EDU</td><td>Gel electrophoresis apparatus</td></tr><tr><td>NaCl</td><td>Thermo Fisher</td><td>S640-3</td><td></td></tr><tr><td>NaOH</td><td>Thermo Fisher</td><td>S318-1</td><td></td></tr><tr><td>Pefabloc SC</td><td>Roche</td><td>11429876001</td><td>Protease inhibitor</td></tr><tr><td>pET42</td><td>EMD Millipore</td><td>70562</td><td>Expression plasmid</td></tr><tr><td>Precision plus all blue standard</td><td>Biorad</td><td>1610373</td><td>Molecular protein standard for SDS-PAGE</td></tr><tr><td>Quickchange mutagenesis kit</td><td>Agilent technologies</td><td>200521</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Thermo Fisher</td><td>BP166</td><td></td></tr><tr><td>Suc-LLVY-AMC</td><td>Enzo lifesciences</td><td>BML P802-0005</td><td>Fluorogenic substrate</td></tr><tr><td>Talon Metal Affinity Resin</td><td>Clontech</td><td>635502</td><td>Immobilized Cobalt Affinity Resin</td></tr><tr><td>TEMED</td><td>Sigma</td><td>T7024</td><td></td></tr><tr><td>Tris</td><td>US Biologicals</td><td>T8600</td><td></td></tr><tr><td>Triton-X100</td><td>Sigma</td><td>93426</td><td></td></tr><tr><td>Tryptone</td><td>Bacto BD</td><td>211699</td><td></td></tr><tr><td>UV sample tray</td><td>Biorad</td><td>1708271</td><td>For UV imaging of gels</td></tr><tr><td>Yeast extract</td><td>Bacto BD</td><td>212720</td><td></td></tr></tbody></table>

examining proteasome assembly with recombinant archaeal proteasomes and nondenaturing page:  the case for a combined approach

蛋白酶在生活的各个领域中。它们提供了细胞内的蛋白质分解的真核生物的主要途径，尽管它们的装配是不完全理解。所有蛋白酶含有一个结构上保守的芯颗粒（CP）或20S蛋白酶，含有夹在两个七聚体α亚基环之间的两个七聚体β亚基环。相比，他们的真核同行古20S蛋白酶体在成分简单，但他们都有着共同的组装机理。因此，古细菌20S的蛋白酶体继续是真核蛋白酶体组件重要的模型。具体来说，再加上非变性聚丙烯酰胺凝胶电泳（PAGE）古20S蛋白酶体的重组表达已经取得了许多重要见解蛋白酶体生物合成。在这里，我们讨论来改善古蛋白酶体α的共表达通常的战略手段和nondenaturiβ亚基前NG PAGE。我们表明，虽然快速，高效，单独共表达的方式可能会错过关键总成的中间体。在蛋白酶体中的情况下，共表达可能不允许检测半蛋白酶体，中间包含一个完整的α型环和一个完整的β型环。然而，该中间体易于通过裂解物混合进行检测。我们建议用裂解液混合相结合共同表达产生一种方法，是在分析装配更彻底，但仍然劳动nonintensive。这种方法可以用于其它重组多蛋白复合物的研究是有用的。

蛋白酶体组件（ 图1）开始时，α亚单位结合形成环9。这可以在从古海藻甲烷球菌 S2α亚基在大肠杆菌中被表达为C末端六组氨酸标签（his标记）衍生物（ 表1）加以说明。当重组体α-他的蛋白质通过ICAR纯化，并分析了非变性PAGE中，观察到（ 图2A，泳道1）两个频带。之前我们已经证实，这些对应的单α环（SR）和双α-戒指（DR）18。 DR是穷途末路的物种，是不是生产为随后的装配9,18。 当α-他的亚基用β亚基共表达，表示由该重组古细菌蛋白酶体的组件被测定时，观察到一个新种的670 kDa的大小支架附近迁移通常的方法ARD（ 图2A，泳道3）。本种是蛋白水解活性的（ 图2B，泳道3）和仅包含完全成熟的β亚基（mβ），其前肽已被删除（ 图2C，第3道）。该品种是完全成熟的CP。此示例还含有一些SR物种，这是预期的，因为SR是公知的装配中间体，但没有DR物种。 α-他当缺乏DR和β亚基共表达表明，正确装配正在发生速度不够快，使得β亚基掺入能够outcompete DR的非生产性形成（更详细的分析见18）。 为了证明共表达方法的限制，并且主张的组合的方法的效用，α-他和β亚基分别在大肠杆菌中表达。以下裂解，可溶性级分混合，并通过ICAR之前纯化的蛋白通过非变性PAGE分析。功能齐全的蛋白酶也通过裂解物混合的方法（ 图2A和图2B，泳道2）中产生，并如预期，也观察到了SR物种。在混合裂解液样品的DR物种的再现表明，一旦形成，β亚基无法可逆拆解;这强调了DR的死胡同性质。有趣的是，一个新的物种也出现了混合裂解液样品中，只迁移CP（称为"半"）所示。最近，我们发现，这个物种相当于半蛋白酶体（αβ7 7）18。在这里说明这一点，一个β亚基突变（R166W）的使用。这种突变破坏β-β环相互作用，导致受损的半蛋白酶二聚体18。由于半蛋白酶是直接前体到CP时，R166W突变应导致半蛋白酶的两个累积DA减少CP的形成。当与α-他和β（R166W）亚基裂解物混合进行，观察到的CP较低水平，并增加了"半"物种的水平（ 图2A，泳道4）。这是与这两个波段的前体 - 产物关系一致，并且印证了"半"物种作为半 - 蛋白酶体的身份。突变样品（泳道4对比泳道2）在半蛋白酶体的速度稍快的迁移可能是由于在R166W突变改变β亚基的质荷比。 相比于细胞内的高蛋白质浓度的可视化裂解物混合期间半蛋白酶的能力，主要是由于在裂解物低得多的蛋白浓度。较低浓度导致更少的高效率（ 即较慢）装配，这使得中间体变得更加填充，从而可检测的。除了半蛋白酶体的外观，<html>一个附加的观察突出裂解物混合期间降低的组装效率:未处理的β亚基，它保留其前肽，成为可检测的（proβ）。由于前肽处理不会发生，直到半蛋白酶二聚化，未成熟proβ形式的外观用半蛋白酶积累水平（比较图2C中，第3道与第2道与第4道）相关。在R166W突变体的情况下，裂解物混合期间前肽处理故障是绝对即使CP少量确实形式。这是因为前肽处理，不仅需要半蛋白酶体二聚化，同时也正确形成β-β环接口23其中R166W突变也买不起。共表达与裂解液混合的更详细的叙述，因为它涉及到重组蛋白酶体装配，都可以在这里找到18。 古尔1"SRC ="/文件/ ftp_upload / 54860 / 54860fig1.jpg"/&gt; 图1:核心粒子（CP）组件的原理示意图。 所述α亚基可以组装成一个单一的α环第一（SR），其充当β亚基的掺入模板。这导致产生半蛋白酶中间体（一半）的从而迅速二聚化。并发二聚，β亚基前肽（未示出）自催化除去从而产生了全功能芯颗粒（CP）。双α-环（DR）可以从SR产生，并不能胜任组装成CP。它们的形成表示在对比生产装配事件（实线箭头）一个非生产性组件途径（虚线箭头）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54860/54860fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2"SRC ="/文件/ ftp_upload / 54860 / 54860fig2.jpg"/&gt; 图2:用于测定蛋白酶体大会相结合的办法。 共表达（C）和裂解物混合（L）被用来从古分枝maripaludis研究重组蛋白酶的装配。蛋白质被固定化钴亲和树脂（ICAR）纯化。 （A，B）纯化的蛋白（10微克）被装载到非变性5 - 10％梯度凝胶。电泳后，肽酶活性是通过用含有荧光肽底物缓冲溶液覆盖在凝胶可视SUC-LLVY-AMC（B）中以染色用胶体考马斯染色试剂（A）中的凝胶之前。黑色箭头表示组装20S核心颗粒（CP），半蛋白酶体中间（一半），双α-环（DR）和单α环（SR）的位置。几个分子大小的标准（以kDa）的迁移右侧表示。 （C） </stron从A g&gt;的纯化蛋白（10微克）也装到15％SDS-PAGE凝胶。电泳后，凝胶用胶体考马斯染色试剂染色。 α-他的亚基和完全成熟（mβ）和未成熟（proβ）β亚基的迁移指示。 25 kDa的分子大小标准的位置如右图所示。星号表示截断α-他的亚基裂解过程中从非特异性蛋白水解产生的片段。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54860/54860fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 质粒 </td><td> 基因型 </td><td> 资源 </td></tr><tr><td> AKB191 </td><td> pET42 PSMA，他的</td><td> Kusmierczyk等人，（2011） </td></tr><tr><td> AKB464 </td><td> pET42 PSMA，他PSMB </td><td> Kusmierczyk等人，（2011） </td></tr><tr><td> AKB946 </td><td> pET42 PSMB </td><td> Panfair等人，（2015） </td></tr><tr><td> AKB952 </td><td> pET42 PSMB（R166W） </td><td> Panfair等人，（2015） </td></tr></tbody></table> 表1: 本研究中使用的细菌质粒。 PSMA是古α亚基基因和PSMB是古β亚基基因。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>缓冲液A </td><td>的50mM HEPES-NaOH洗涤，pH值7.5，300mM NaCl的，5mM的MgCl 2的。 </td><td>缓冲器B，C和E，是缓冲含有咪唑衍生物。从在水中制备并储存在黑暗中2M的库存添加咪唑是有用的。 </td></tr><tr><td>本地缓冲解决</td><td> 375毫的Tris-HCl，pH值8.8，0.1％（体积/体积）四甲基乙二胺（TEMED）和0.1％（重量/体积）铵Perulfate（APS）。 </td><td>准备新鲜不超过一个小时前，凝胶进行聚合保持在冰上。当在本地解决缓冲器制备丙烯酰胺凝胶溶液，是有用的，从一个4X库存（1.5M的Tris-HCl，pH值8.8）中添加的Tris-HCl。在APS中加入之前立即聚合。 </td></tr><tr><td>本机运行缓冲液10X股票</td><td> 250毫米的Tris，1.92甘氨酸，不调整pH值。 </td><td></td></tr><tr><td> 5X本土样品缓冲液</td><td>的0.5M的Tris-HCl，pH值8.8，50％（V / V）甘油，溴酚蓝的痕迹。 </td><td>迹线指的是一个非常小的量，通常是通过刮刀转移几粒。 </td></tr><tr><td> 5X SDS样品缓冲</td><td>的0.3M的Tris-HCl，pH值6.8，600mM的二硫苏糖醇（DTT），10％（重量/体积）SDS，50％（体积/体积）甘油和溴酚蓝的痕迹。 </td><td>迹线指的是一个非常小的量，通常是通过刮刀转移几粒。 </td></tr><tr><td>显影缓冲器</td><td>的50mM的Tris-HCl，pH值7.5，5mM的MgCl 2的，1毫摩尔ATP，50mM的SUC-LLVY-AMC。 </td><td> SUC-LLVY-AMC是用于测定蛋白酶活性的荧光肽底物。 </td></tr></tbody></table> 表2:解决方案，在这项研究中使用的介质，和缓冲液。

Watch this Scientific Journal Video about Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE:  The Case for a Combined Approach at JoVE.com

Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE:  The Case for a Combined Approach

该协议使用两个亚基共表达和postlysis亚基重组蛋白酶组件的更彻底的检查混合。

检查蛋白酶体大会与重组古菌蛋白酶体和非变性PAGE:该案例相结合的办法

Proteasomes are found in all domains of life. They provide the major route of intracellular protein degradation in eukaryotes, though their assembly is ...

examining-proteasome-assembly-with-recombinant-archaeal-proteasomes

Nanyang Technological University

Research

JoVE Journal

Biochemistry

6.6K 次观看.  Indiana University-Purdue University, Indianapolis (IUPUI). 该协议使用两个亚基共表达和postlysis亚基重组蛋白酶组件的更彻底的检查混合。 

视频: Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE:  The Case for a Combined Approach

Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.

This article describes a simple and rapid protocol to evaluate the oligomeric state of the dynamin-like GTPase MxA protein from lysates of human cells using a combination of non-denaturing PAGE with western blot analysis.

使用非变性聚丙烯酰胺凝胶电泳人的MxA多亚基蛋白复合物的表征

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral ...

科研

生物化学

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by networks of interacting protein complexes, which are often difficult to investigate because their binding is non-covalent and easily perturbed by purification techniques. This work describes a method of stabilizing and separating native protein complexes from unmodified tissue using two-dimensional polyacrylamide gel electrophoresis. Tissue lysate is loaded onto a non-denaturing blue-native polyacrylamide gel, then an electric current is applied until the protein migrates a short distance into the gel. The gel strip containing the migrated protein is then excised and incubated with the amine-reactive cross-linking reagent dithiobis(succinimidyl propionate), which covalently stabilizes protein complexes. The gel strip containing cross-linked complexes is then cast into a sodium dodecyl sulfate polyacrylamide gel, and the complexes are separated completely. The method relies on techniques and materials familiar to most molecular biologists, meaning it is inexpensive and easy to learn. While it is limited in its ability to adequately separate extremely large complexes, and has not been universally successful, the method was able to capture a wide variety of well-studied complexes, and is likely applicable to many systems of interest.

A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented.

多聚-PAGE:生物样品中的用于拍摄方法和解决蛋白复合体

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, they are important drug targets. We have developed a magnetic-agarose-bead-based assay platform for the investigation of proteolytic activity, which is based on the use of recombinant fusion protein substrates. In order to demonstrate the use of this assay system, a protocol is presented on the example of human immunodeficiency virus type 1 (HIV-1) protease. The introduced assay platform can be utilized efficiently in the biochemical characterization of proteases, including enzyme activity measurements in mutagenesis, kinetic, inhibition, or specificity studies, and it may be suitable for high-throughput substrate screening or may be adapted to other proteolytic enzymes.
In this assay system, the applied substrates contain N-terminal hexahistidine (His6) and maltose-binding protein (MBP) tags, cleavage sites for tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein. The substrates can be efficiently produced in Escherichia coli cells and easily purified using nickel (Ni)-chelate-coated beads. During the assay, the proteolytic cleavage of bead-attached substrates leads to the release of fluorescent cleavage fragments, which can be measured by fluorimetry. Additionally, cleavage reactions can be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A protocol for the in-gel renaturation of assay components is also described, as partial renaturation of fluorescent proteins enables their detection based on molecular weight and fluorescence.

Here, we present the detailed procedure of a recently developed protease assay platform utilizing N-terminal hexahistidine/maltose-binding protein and fluorescent protein-fused recombinant substrates attached to the surface of nickel-nitrilotriacetic acid magnetic agarose beads. A subsequent in-gel analysis of the assay samples separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is also presented.

重组融合蛋白在荧光蛋白酶检测平台中的应用及其凝胶内再生

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, ...

Hybrid Clear/Blue Native Electrophoresis for the Separation and Analysis of Mitochondrial Respiratory Chain Supercomplexes

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer structural and functional advantages to mitochondria. SCs have been identified in many species, from yeast to mammal, and an increasing number of studies report disruption of their organization in genetic and acquired human diseases. As a result, an increasing number of laboratories are interested in analyzing SCs, which can be methodologically challenging. This article presents an optimized protocol that combines the advantages of Blue- and Clear-Native PAGE methods to resolve and analyze SCs in a time-effective manner. With this hybrid CN/BN-PAGE method, mitochondrial SCs extracted with optimal amounts of the mild detergent digitonin are exposed briefly to the anionic dye Coomassie Blue (CB) at the beginning of the electrophoresis, without exposure to other detergents. This short exposure to CB allows to separate and resolve SCs as effectively as with traditional BN-PAGE methods, while avoiding the negative impact of high CB levels on in-gel activity assays, and labile protein-protein interactions within SCs. With this protocol it is thus possible to combine precise and rapid in gel activity measurements with analytical techniques involving 2D electrophoresis, immuno-detection, and/or proteomics for advanced analysis of SCs.

Here we present a protocol to extract, resolve and identify mitochondrial supercomplexes which minimizes exposure to detergents and Coomassie Blue. This protocol offers an optimal balance between resolution, and preservation of enzyme activities, while minimizing the risk of losing labile protein-protein interactions.

混合光/蓝色原生电泳在线粒体呼吸链超综合体分离与分析中的作用

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

蓝原生聚丙烯酰胺凝胶电泳（BN - PAGE）分析，从细胞裂解液多蛋白复合物

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

生物学

Assaying Proteasomal Degradation in a Cell-free System in Plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.
The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.

Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular &#8220;tags&#8221; for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

测定蛋白酶体降解的植物无细胞系统

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

测定法错误折叠的蛋白质在细胞中的降解

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...

Combining Chemical Cross-linking and Mass Spectrometry of Intact Protein Complexes to Study the Architecture of Multi-subunit Protein Assemblies

Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is therefore fundamental for the understanding of cellular processes. However, many protein complexes are dynamic assemblies and are not accessible by conventional structural techniques. Mass spectrometry contributes to the structural investigation of these assemblies, and particularly the combination of various mass spectrometric techniques delivers valuable insights into their structural arrangement.
In this article, we describe the application and combination of two complementary mass spectrometric techniques, namely chemical cross-linking coupled with mass spectrometry and native mass spectrometry. Chemical cross-linking involves the covalent linkage of amino acids in close proximity by using chemical reagents. After digestion with proteases, cross-linked di-peptides are identified by mass spectrometry and protein interactions sites are uncovered. Native mass spectrometry on the other hand is the analysis of intact protein assemblies in the gas phase of a mass spectrometer. It reveals protein stoichiometries as well as protein and ligand interactions. Both techniques therefore deliver complementary information on the structure of protein-ligand assemblies and their combination proved powerful in previous studies.

The architecture of protein complexes is essential for their function. Combining various mass spectrometric techniques proved powerful to study their assembly. We provide protocols for chemical cross-linking and native mass spectrometry and show how these complementary techniques help to elucidate the architecture of multi-subunit protein assemblies.

完整蛋白质复合物的化学交联和质谱分析研究多亚基蛋白质组合体的体系结构

Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is ...

Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes

Sampling the phloem of higher plants is often laborious and significantly dependent on the plant species. However, proteome studies under denaturing conditions could be achieved in different plant species. Native protein:protein and protein:nucleic acid complexes from phloem samples have as yet scarcely been analyzed, although they might play important roles in maintenance of this specialized compartment or in long-distance signaling. Large molecular assemblies can be isolated using a blue native gel electrophoresis (BN-PAGE). Their protein components can be separated by a subsequent sodium dodecyl sulfate PAGE (SDS-PAGE). However, proteins with similar molecular weights co-migrate, what can hinder protein identification by mass spectrometry. Combining BN-PAGE with two different denaturing gel electrophoresis steps, namely Tris-Tricine-urea and SDS-PAGE, enables the additional separation of proteins according to their hydrophilicity/hydrophobicity and thus increases resolution and the success of protein identification. It even allows distinguishing proteins that only differ in their posttranslational modifications. In addition, blue native northern blotting can be applied to identify the RNA components in macromolecular complexes. We show that our protocol is suitable to unravel the protein and RNA components of native protein:protein and ribonucleoprotein (RNP) complexes occurring in phloem samples. Combining a blue native PAGE with two different denaturing PAGE steps can help to separate different kinds of large protein complexes, and also enables an increased identification rate of their components by mass spectrometry. Furthermore, the protocol is robust enough to simultaneously detect potentially bound nucleic acids within single protein complexes.