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>

<ol>
	<li>Wan, C., 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=Panorama+of+ancient+metazoan+macromolecular+complexes.">Panorama of ancient metazoan macromolecular complexes.</a> Nature. 525, 339-344 (2015).</li><li>Marsh, J. A., Teichmann, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure,+dynamics,+assembly,+and+evolution+of+protein+complexes.">Structure, dynamics, assembly, and evolution of protein complexes.</a> Annu Rev Biochem. 84, 551-575 (2015).</li><li>Williamson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperativity+in+macromolecular+assembly.">Cooperativity in macromolecular assembly.</a> Nat Chem Biol. 4, 458-465 (2008).</li><li>Finley, D., Ulrich, H. D., Sommer, T., Kaiser, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ubiquitin-proteasome+system+of+Saccharomyces+cerevisiae.">The ubiquitin-proteasome system of Saccharomyces cerevisiae.</a> Genetics. 192, 319-360 (2012).</li><li>Groll, M., 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=Structure+of+20S+proteasome+from+yeast+at+2.4+A+resolution.">Structure of 20S proteasome from yeast at 2.4 A resolution.</a> Nature. 386, 463-471 (1997).</li><li>Lander, G. C., 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=Complete+subunit+architecture+of+the+proteasome+regulatory+particle.">Complete subunit architecture of the proteasome regulatory particle.</a> Nature. 482, 186-191 (2012).</li><li>Lowe, J., 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=Crystal+structure+of+the+20S+proteasome+from+the+archaeon+T.+acidophilum+at+3.4+A+resolution.">Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 A resolution.</a> Science. 268, 533-539 (1995).</li><li>Hu, G., 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=Structure+of+the+Mycobacterium+tuberculosis+proteasome+and+mechanism+of+inhibition+by+a+peptidyl+boronate.">Structure of the Mycobacterium tuberculosis proteasome and mechanism of inhibition by a peptidyl boronate.</a> Mol Microbiol. 59, 1417-1428 (2006).</li><li>Zwickl, P., Kleinz, J., Baumeister, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+elements+in+proteasome+assembly.">Critical elements in proteasome assembly.</a> Nat Struct Biol. 1, 765-770 (1994).</li><li>Groll, M., Brandstetter, H., Bartunik, H., Bourenkow, G., Huber, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigations+on+the+maturation+and+regulation+of+archaebacterial+proteasomes.">Investigations on the maturation and regulation of archaebacterial proteasomes.</a> J Mol Biol. 327, 75-83 (2003).</li><li>Frankenberg, R. J., Hsu, T. S., Yakota, H., Kim, R., Clark, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+denaturation+and+elevated+folding+temperatures+are+required+for+wild-type+activity+and+stability+of+recombinant+Methanococcus+jannaschii+20S+proteasome.">Chemical denaturation and elevated folding temperatures are required for wild-type activity and stability of recombinant Methanococcus jannaschii 20S proteasome.</a> Protein Sci. 10, 1887-1896 (2001).</li><li>Maupin-Furlow, J. A., Aldrich, H. C., Ferry, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+characterization+of+the+20S+proteasome+from+the+methanoarchaeon+Methanosarcina+thermophila.">Biochemical characterization of the 20S proteasome from the methanoarchaeon Methanosarcina thermophila.</a> J Bacteriol. 180, 1480-1487 (1998).</li><li>Maupin-Furlow, J. A., Ferry, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+proteasome+from+the+methanogenic+archaeon+Methanosarcina+thermophila.">A proteasome from the methanogenic archaeon Methanosarcina thermophila.</a> J Biol Chem. 270, 28617-28622 (1995).</li><li>Wittig, I., Braun, H. P., Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+PAGE.">Blue native PAGE.</a> Nat Protoc. 1, 418-428 (2006).</li><li>Langer, T., Pfeifer, G., Martin, J., Baumeister, W., Hartl, F. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chaperonin-mediated+protein+folding:+GroES+binds+to+one+end+of+the+GroEL+cylinder,+which+accommodates+the+protein+substrate+within+its+central+cavity.">Chaperonin-mediated protein folding: GroES binds to one end of the GroEL cylinder, which accommodates the protein substrate within its central cavity.</a> EMBO J. 11, 4757-4765 (1992).</li><li>McKenzie, M., Lazarou, M., Thorburn, D. R., Ryan, M. 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

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.