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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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+mitochondrial+subunit+assembly+into+respiratory+chain+complexes+using+Blue+Native+polyacrylamide+gel+electrophoresis.">Analysis of mitochondrial subunit assembly into respiratory chain complexes using Blue Native polyacrylamide gel electrophoresis.</a> Anal Biochem. 364, 128-137 (2007).</li><li>Tomko, R. J., Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+the+Rpn12+subunit+couples+completion+of+proteasome+regulatory+particle+lid+assembly+to+lid-base+joining.">Incorporation of the Rpn12 subunit couples completion of proteasome regulatory particle lid assembly to lid-base joining.</a> Mol Cell. 44, 907-917 (2011).</li><li>Panfair, D., Ramamurthy, A., Kusmierczyk, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-ring+Independent+Assembly+of+the+20S+Proteasome.">Alpha-ring Independent Assembly of the 20S Proteasome.</a> Sci Rep. 5, 13130 (2015).</li><li>Zimmerman, S. B., Trach, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+macromolecule+concentrations+and+excluded+volume+effects+for+the+cytoplasm+of+Escherichia+coli.">Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli.</a> J Mol Biol. 222, 599-620 (1991).</li><li>Kusmierczyk, A. R., Kunjappu, M. J., Kim, R. Y., Hochstrasser, 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+conserved+20S+proteasome+assembly+factor+requires+a+C-terminal+HbYX+motif+for+proteasomal+precursor+binding.">A conserved 20S proteasome assembly factor requires a C-terminal HbYX motif for proteasomal precursor binding.</a> Nat Struct Mol Biol. 18, 622-629 (2011).</li><li>Smith, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Anal Biochem. 150, 76-85 (1985).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227, 680-685 (1970).</li><li>Chen, P., Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocatalytic+subunit+processing+couples+active+site+formation+in+the+20S+proteasome+to+completion+of+assembly.">Autocatalytic subunit processing couples active site formation in the 20S proteasome to completion of assembly.</a> Cell. 86, 961-972 (1996).</li><li>Li, X., Kusmierczyk, A. R., Wong, P., Emili, A., Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-Subunit+appendages+promote+20S+proteasome+assembly+by+overcoming+an+Ump1-dependent+checkpoint.">beta-Subunit appendages promote 20S proteasome assembly by overcoming an Ump1-dependent checkpoint.</a> EMBO J. 26, 2339-2349 (2007).</li><li>Kusmierczyk, A. R., Kunjappu, M. J., Funakoshi, M., Hochstrasser, 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+multimeric+assembly+factor+controls+the+formation+of+alternative+20S+proteasomes.">A multimeric assembly factor controls the formation of alternative 20S proteasomes.</a> Nat Struct Mol Biol. 15, 237-244 (2008).</li></ol>

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...

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。...

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

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

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

从外膜囊泡内蛋白质导演包装<em&gt;大肠杆菌</em&gt;:设计，生产和纯化

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

科研

生物化学

Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, particle size and shape, and surface-to-volume ratio of macromolecules. The technique is applicable to a very wide variety of solution conditions spanning a broad range of concentrations, pH values, ionic strengths, temperatures, additives, etc., but the sample is required to be monodisperse. This caveat led to the implementation of liquid chromatography systems on SAXS beamlines. Here, we describe the upstream integration of size-exclusion (SEC) and ion-exchange chromatography (IEC) on a beamline, different methods for optimal background subtraction, and data reduction. As an example, we describe how we use SEC- and IEC-SAXS on a fragment of the essential vaccinia virus protein D5, consisting of a D5N helicase domain. We determine its overall shape and molecular weight, showing the hexameric structure of the protein.

The determination of the solution structure of a protein by small angle X-ray scattering (SAXS) requires monodisperse samples. Here, we present two possibilities to ensure minimal delays between sample preparation and data acquisition: online size-exclusion chromatography (SEC) and online ion-exchange chromatography (IEC).

在线体积排阻和离子交换色谱法SAXS光束线

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, ...

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 ...

Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications (PTMs). They are newly identified species with unknown means of establishment and functional implications. Current analytical methods are inadequate to detect the copy-specific occurrence of PTMs on the nucleosomal sister histones. This protocol presents a biochemical method for the in vitro reconstitution of nucleosomes containing differentially isotope-labeled sister histones. The generated complex can be also asymmetrically modified, after including a premodified histone pool during refolding of histone subcomplexes. These asymmetric nucleosome preparations can be readily reacted with histone-modifying enzymes to study modification cross-talk mechanisms imposed by the asymmetrically pre-incorporated PTM using nuclear magnetic resonance (NMR) spectroscopy. Particularly, the modification reactions in real-time can be mapped independently on the two sister histones by performing different types of NMR correlation experiments, tailored for the respective isotope type. This methodology provides the means to study crosstalk mechanisms that contribute to the formation and propagation of asymmetric PTM patterns on nucleosomal complexes.

This protocol describes the reconstitution of nucleosomes containing differentially isotope-labeled sister histones. At the same time, asymmetrically post-translationally modified nucleosomes can be generated after using a premodified histone copy. These preparations can be readily used to study modification crosstalk mechanisms, simultaneously on both sister histones, by using high-resolution NMR spectroscopy.

核小体的重建与差异同位素标记的姐妹组蛋白

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications ...

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the disordered and protease labile C-terminus of the protein. This manuscript describes the methods to purify recombinant Nsa1 from S. cerevisiae for structural analysis by both X-ray crystallography and SAXS. X-ray crystallography was utilized to solve the structure of the well-ordered N-terminal WD40 domain of Nsa1, and then SAXS was used to resolve the structure of the C-terminus of Nsa1 in solution. Solution scattering data was collected from full-length Nsa1 in solution. The theoretical scattering amplitudes were calculated from the high-resolution crystal structure of the WD40 domain, and then a combination of rigid body and ab initio modeling revealed the C-terminus of Nsa1. Through this hybrid approach the quaternary structure of the entire protein was reconstructed. The methods presented here should be generally applicable for the hybrid structural determination of other proteins composed of a mix of structured and unstructured domains.

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

This method describes the cloning, expression, and purification of recombinant Nsa1 for structural determination by X-ray crystallography and small-angle X-ray scattering (SAXS), and is applicable for the hybrid structural analysis of other proteins containing both ordered and disordered domains.

结合 x 射线晶体学与小角度 x 射线散射到模型的非结构化区域的 Nsa1 从s 酿酒酵母

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the ...

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, ...

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.

Here, we present a protocol to conjugate protein monomer by enzymes forming protein polymer with a controlled sequence and immobilize it on the surface for single-molecule force spectroscopy studies.

OaAEP1介导酶合成和聚合蛋白固定，用于单分子力光谱

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

生物学

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often detrimental to protein folding and, in particular, chromophore maturation in green fluorescent proteins and related variants. A reasonable alternative is to manipulate the translation of the protein so that all Pro residues are replaced residue-specifically by analogs, a method known as selective pressure incorporation (SPI). The built-in chemical modifications can be used as a kind of &#34;molecular surgery&#34; to finely dissect measurable changes or even rationally manipulate different protein properties. Here, the study demonstrates the usefulness of the SPI method to study the role of prolines in the organization of the typical &#946;-barrel structure of spectral variants of the green fluorescent protein (GFP) with 10-15 prolines in their sequence: enhanced green fluorescent protein (EGFP), NowGFP, and KillerOrange. Pro residues are present in connecting sections between individual &#946;-strands and constitute the closing lids of the barrel scaffold, thus being responsible for insulation of the chromophore from water, i.e., fluorescence properties. Selective pressure incorporation experiments with (4R)-fluoroproline (R-Flp), (4S)-fluoroproline (S-Flp), 4,4-difluoroproline (Dfp), and 3,4-dehydroproline (Dhp) were performed using a proline-auxotrophic E. coli strain as expression host. We found that fluorescent proteins with S-Flp and Dhp are active (i.e., fluorescent), while the other two analogs (Dfp and R-Flp) produced dysfunctional, misfolded proteins. Inspection of UV-Vis absorption and fluorescence emission profiles showed few characteristic alterations in the proteins containing Pro analogs. Examination of the folding kinetic profiles in EGFP variants showed an accelerated refolding process in the presence of S-Flp, while the process was similar to wild-type in the protein containing Dhp. This study showcases the capacity of the SPI method to produce subtle modifications of protein residues at an atomic level (&#34;molecular surgery&#34;), which can be adopted for the study of other proteins of interest. It illustrates the outcomes of proline replacements with close chemical analogs on the folding and spectroscopic properties in the class of &#946;-barrel fluorescent proteins.

To overcome the limitations of classical site-directed mutagenesis, proline analogs with specific modifications were incorporated into several fluorescent proteins. We show how the replacement of hydrogen by fluorine or of the single by double bonds in proline residues ("molecular surgery") affects fundamental protein properties, including their folding and interaction with light.

荧光蛋白中脯氨酸类似物对脯氨酸的残基特异换:骨架的"分子手术"如何影响折叠和稳定性

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often ...

生物工程

The Proteasome Structure

The ubiquitin-proteasome pathway is a well-known mechanism utilized by eukaryotic cells to remove cytoplasmic proteins that are misfolded, damaged, or no longer needed. In this pathway, the protein that needs to be eliminated undergoes a process called ubiquitination, where a chain of ubiquitin molecules is attached to the 48th lysine residue of the target protein. This ubiquitin modification helps the proteasome distinguish between a target protein and a healthy protein.
The proteasome is an ATP-driven, large multi-subunit protease (26S) found in the nucleus and cytoplasm of all eukaryotes. The 26S proteasome comprises a central hollow cylindrical core (the 20S) and ring-shaped protein caps (19S). The 20S core particle is barrel-shaped, containing stacked rings with proteolytic active sites lined inside. The 19S caps act as gatekeepers and regulatory units of the proteasome. They are present at one or both ends of the core.
Caps have ubiquitin receptors, ubiquitin hydrolase, and unfoldase, each with a unique function. The ubiquitin receptor recognizes and allows ubiquitin-tagged proteins into the core, the hydrolase deubiquitinase cleaves ubiquitin from the substrate, and the unfoldase protein unfolds target proteins as they enter the protein core.
The hexameric unfoldases create a ring structure in the proteasome cap and belong to a large class of AAA proteins. Their mechanistic action is similar to other ATP-dependent helicases that cause DNA unwinding. The ATP-bound unfoldase ring binds a folded target protein held in place by its ubiquitin tag at the proteasomal cap. Next, a conformational change, driven by ATP hydrolysis, pulls the target protein substrate into the 20S core and strains the ring structure. As a result, when a protein is subjected to pulling forces, it can undergo two possible outcomes&#8212; it may either undergo partial unfolding and enter the cylindrical core, or it can retain its structure while partially retracting.
Highly stable substrates generally require hundreds of cycles of ATP hydrolysis before being pulled through the AAA ring. Once de-ubiquitylated and unfolded, the target protein is exposed to proteases inside the proteasomal core for digestion. In contrast to free cellular proteases, which quickly dissociate after their action on the target protein, the proteasome core holds the target protein until it is fully digested into smaller peptides. These small peptides are then released into the cytosol for further processing.&#160;

蛋白酶体结构

The ubiquitin-proteasome pathway is a well-known mechanism utilized by eukaryotic cells to remove cytoplasmic proteins that are misfolded, damaged, or no ...