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...

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 border="0" cellpadding="0" cellspacing="0" width="701">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:165px;">Acrylamide (40%) solution</td>
			<td style="width:123px;">Biorad</td>
			<td style="width:105px;">1610104</td>
			<td style="width:309px;">Unpolymerized acrylamide is a neurotoxin. Wear proper protective equipment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">Amicon ultra 0.5ml centrifugal filters</td>
			<td style="width:123px;">EMDMillipore</td>
			<td>UFC501024</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">Ammonium persulfate</td>
			<td>Sigma</td>
			<td>A3678</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">ATP</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:105px;">A7699</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">BCA assay kit</td>
			<td style="width:123px;">Pierce</td>
			<td style="width:105px;">23225</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">Bisacrylamide (2%) solution</td>
			<td style="width:123px;">Biorad</td>
			<td style="width:105px;">1610142</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Bromophenol blue</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:105px;">B8026</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">DNaseI</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:105px;">DN25</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Dithiothreitol (DTT)</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:105px;">BP172</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">E.coli BL21 competent cells</td>
			<td style="width:123px;">EMD Millipore</td>
			<td style="width:105px;">69450</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">GelCode Blue</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:105px;">24592</td>
			<td style="width:309px;">Colloidal coomassie stain reagent for gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Gel doc EZ system</td>
			<td style="width:123px;">Biorad</td>
			<td>1708270</td>
			<td style="width:309px;">Gel documentation system</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:165px;">Gel releasers</td>
			<td style="width:123px;">Biorad</td>
			<td>1653320</td>
			<td style="width:309px;">Wedge shaped plastic used to separate gel plates; useful for spreading liquid.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Glass rod</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td>11-380B</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Glycerol</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:105px;">49767</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Glycine</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:105px;">BP3865</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Hamilton syringe</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td>14-813-38</td>
			<td style="width:309px;">Glass syringe for loading gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">HEPES</td>
			<td style="width:123px;">US Biologicals</td>
			<td style="width:105px;">H2010</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">HMW Native calibration kit</td>
			<td style="width:123px;">GE Healthcare</td>
			<td style="width:105px;">170445-01</td>
			<td style="width:309px;">High molecular weight protein standards</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Hoefer SG30</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:105px;">03-500-277</td>
			<td style="width:309px;">Gradient maker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Imidazole</td>
			<td style="width:123px;">US Biologicals</td>
			<td style="width:105px;">280671</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">IPTG</td>
			<td style="width:123px;">US Biologicals</td>
			<td style="width:105px;">I8500</td>
			<td style="width:309px;">For induction of protein expression</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Isopropanol</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:105px;">BP26181</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Kanamycin sulfate</td>
			<td style="width:123px;">US Biologicals</td>
			<td style="width:105px;">K0010</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Lysozyme</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:105px;">L6876</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:165px;">MgCl2</td>
			<td style="width:123px;">Fluka analytical</td>
			<td style="width:105px;">630680</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Mini Protean Tetra Cell</td>
			<td style="width:123px;">Biorad</td>
			<td style="width:105px;">1658002EDU</td>
			<td style="width:309px;">Gel electrophoresis apparatus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">NaCl</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:105px;">S640-3</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">NaOH</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:105px;">S318-1</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Pefabloc SC</td>
			<td style="width:123px;">Roche</td>
			<td style="width:105px;">11429876001</td>
			<td style="width:309px;">Protease inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">pET42</td>
			<td style="width:123px;">EMD Millipore</td>
			<td style="width:105px;">70562</td>
			<td style="width:309px;">Expression plasmid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">Precision plus all blue standard</td>
			<td style="width:123px;">Biorad</td>
			<td style="width:105px;">1610373</td>
			<td style="width:309px;">Molecular protein standard for SDS-PAGE</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">Quickchange mutagenesis kit</td>
			<td style="width:123px;">Agilent technologies</td>
			<td style="width:105px;">200521</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">Sodium dodecyl sulfate (SDS)</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:105px;">BP166</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">Suc-LLVY-AMC</td>
			<td style="width:123px;">Enzo lifesciences</td>
			<td style="width:105px;">BML P802-0005</td>
			<td style="width:309px;">Fluorogenic substrate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">Talon Metal Affinity Resin</td>
			<td style="width:123px;">Clontech</td>
			<td style="width:105px;">635502</td>
			<td style="width:309px;">Immobilized-cobalt affinity resin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">TEMED</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:105px;">T7024</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Tris</td>
			<td style="width:123px;">US Biologicals</td>
			<td style="width:105px;">T8600</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Triton-X100</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:105px;">93426</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Tryptone</td>
			<td style="width:123px;">Bacto BD</td>
			<td style="width:105px;">211699</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">UV sample tray</td>
			<td style="width:123px;">Biorad</td>
			<td>1708271</td>
			<td style="width:309px;">For UV imaging of gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Yeast extract</td>
			<td style="width:123px;">Bacto BD</td>
			<td style="width:105px;">212720</td>
			<td style="width:309px;"></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。...

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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 ...

The overall goal of this experiment is to analyze assembly of a multi-protein complex such as the proteasome, using recombinant subunits, and nondenaturing Polyacrylamide Gel Electrophoresis, or PAGE.This method can help answer key questions in the proteasome field such as, what intermediate species are encountered along the assembly pathway of this protein complex.The main advantage of this technique is that it allows rapid screening of assembly by two parallel approaches, allowing detection of on and off pathway intermediates.This procedure begins with bacterial expression as described in the text protocol.To perform bacterial lysis, thaw the induced cell pallet co-expressing the desired combination of proteasome subunits, on ice for five minutes.Once thawed, resuspend the pallet in 600 microliters of Lysis Buffer.Incubate the suspension at 30

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

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

6.6K 次观看.  Indiana University-Purdue University, Indianapolis (IUPUI). The overall goal of this experiment is to analyze assembly of a multi-protein complex such as the proteasome, using recombinant subunits, and nondenaturing Polyacrylamide Gel Electrophoresis, or PAGE.This method can help answer key questions in the proteasome field such as, what intermediate species are encountered along the assembly pathway of this protein complex.The main advantage of this technique is that it allows rapid screening of assembly by two parallel approaches, allowing detection...

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

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

可逆二硫键交联胶束生产的简便和有效途径

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

科研

生物化学

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

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 ...

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of the kinase activity of LRRK2 has been implicated in the pathogenesis of PD, it is essential to establish a method to evaluate the physiological levels of the kinase activity of LRRK2. Recent studies revealed that LRRK2 phosphorylates members of the Rab GTPase family, including Rab10, under physiological conditions. Although the phosphorylation of endogenous Rab10 by LRRK2 in cultured cells could be detected by mass spectrometry, it has been difficult to detect it by immunoblotting due to the poor sensitivity of currently available phosphorylation-specific antibodies for Rab10. Here, we describe a simple method of detecting the endogenous levels of Rab10 phosphorylation by LRRK2 based on immunoblotting utilizing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with a phosphate-binding tag (P-tag), which is N-(5-(2-aminoethylcarbamoyl)pyridin-2-ylmetyl)-N,N&#39;,N&#39;-tris(pyridin-2-yl-methyl)-1,3-diaminopropan-2-ol. The present protocol not only provides an example of the methodology utilizing the P-tag but also enables the assessment of how mutations as well as inhibitor treatment/administration or any other factors alter the downstream signaling of LRRK2 in cells and tissues.

The present study describes a simple method of detecting endogenous levels of Rab10 phosphorylation by leucine-rich repeat kinase 2.

用磷酸盐结合标记的 SDS-页 Rab10 磷酸化检测 LRRK2 活性

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of ...

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to precisely maneuver the nucleation and crystal growth towards desired sizes of protein crystals. The controlled crystallization is based on sample investigation with in situ Dynamic Light Scattering (DLS) while all visual changes in the droplet are monitored online with the help of a microscope coupled to a CCD camera, thus enabling a full investigation of the protein droplet during all stages of crystallization. The use of in situ DLS measurements throughout the entire experiment allows a precise identification of the highly supersaturated protein solution transitioning to a new phase &#8211; the formation of crystal nuclei. By identifying the protein nucleation stage, the crystallization can be optimized from large protein crystals to the production of protein microcrystals. The experimental protocol shows an interactive crystallization approach based on precise automated steps such as precipitant addition, water evaporation for inducing high supersaturation, and sample dilution for slowing induced homogeneous nucleation or reversing phase transitions.

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Here we present a protocol for controlled production of protein microcrystals. The process uses an automated device allowing controlled manipulation of several crystallization parameters. The protein crystallization is carried-out by controlled and automated addition of crystallization solutions while monitoring and investigating the radius distribution of particles in the crystallization droplet.

采用自动结晶耦合原位动态光散射的不同尺寸生长蛋白晶体

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

洗涤剂辅助重组重组果蝇阿拉他素到脂质混合测定脂质体

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall process costs. Alternative, cost-effective capture steps are therefore valuable for industrial-scale manufacturing, where large quantities of a single mAb are produced. Here we present a method for the immobilization of a DsRed-based epitope ligand to a cross-linked agarose resin allowing the selective capture of the HIV-neutralizing antibody 2F5 from crude plant extracts without using Protein A. The linear epitope ELDKWA was first genetically fused to the fluorescent protein DsRed and the fusion protein was expressed in transgenic tobacco (Nicotiana tabacum) plants before purification by immobilized metal-ion affinity chromatography. Furthermore, a method based on activated cross-linked agarose was optimized for high ligand density, efficient coupling and low costs. The pH and buffer composition and the soluble ligand concentration were the most important parameters during the coupling procedure, which was improved using a design-of-experiments approach. The resulting affinity resin was tested for its ability to selectively bind the target mAb in a crude plant extract and the elution buffer was optimized for high mAb recovery, product activity and affinity resin stability. The method can easily be adapted to other antibodies with linear epitopes. The new resins allow gentler elution conditions than Protein A and could also reduce the costs of an initial capture step for mAb production.

In this procedure, a DsRed-based epitope ligand is immobilized to produce a highly selective affinity resin for the capture of monoclonal antibodies from crude plant extracts or cell culture supernatants, as an alternative to Protein A.

活联甘蔗，用于亲和色谱树脂的快速发展 - 抗体捕获作为案例研究

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...

Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the homeostasis of adult tissues. Interestingly, oxidative stress can alter these processes, thus contributing to the pathophysiology of diseases such as metastatic cancer. Therefore, understanding the mechanism(s) of how cells attach and spread on the ECM during perturbations in redox status can provide insight into normal and disease states. Described below is a step-wise protocol that utilizes an immunofluorescence-based assay to specifically quantify cell adhesion and spreading of immortalized fibroblast cells on fibronectin (FN) in vitro. Briefly, anchorage-dependent cells are held in suspension and exposed to the ATM kinase inhibitor Ku55933 to induce oxidative stress. Cells are then plated on FN-coated surface and allowed to attach for predetermined periods of time. Cells that remain attached are fixed and labeled with fluorescence-based antibody markers of adhesion (e.g., paxillin) and spreading (e.g., F-actin). Data acquisition and analysis are performed using commonly available laboratory equipment, including an epifluorescence microscope and freely available Fiji software. This procedure is highly versatile and can be modified for a variety of cell lines, ECM proteins, or inhibitors in order to examine a broad range of biological questions.

This method is useful for quantifying the early dynamics of cellular adhesion and spreading of anchorage-dependent cells onto the fibronectin. Furthermore, this assay can be used to investigate the effects of altered redox homeostasis on cell spreading and/or cell adhesion-related intracellular signaling pathways.

在氧化应激期间细胞粘附和纤维蛋白扩散动力学

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the ...

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be used to generate different diterpenoid scaffolds. Purified compounds can be used in various downstream applications, such as metabolite structural analyses, enzyme structure-function studies, and in vitro and in planta bioactivity experiments.

Here we present easy to use protocols for producing and purifying diterpenoid metabolites through the combinatorial expression of biosynthetic enzymes in Escherichia coli or Nicotiana benthamiana, followed by chromatographic product purification. The resulting metabolites are suitable for various studies including molecular structure characterization, enzyme functional studies, and bioactivity assays.

一种可定制的酶化生产与纯化天然产物的方法

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological ...

Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein

Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human cancers, is processed by farnesylation and carboxymethylation due to the presence of a &#39;CAAX&#39; box motif at the C-terminus. An engineered baculovirus system was used to express farnesylated and carboxymethylated KRAS4b in insect cells and has been described previously. Here, we describe the detailed, practical purification and biochemical characterization of the protein. Specifically, affinity and ion exchange chromatography were used to purify the protein to homogeneity. Intact and native mass spectrometry was used to validate the correct modification of KRAS4b and to verify nucleotide binding. Finally, membrane association of farnesylated and carboxymethylated KRAS4b to liposomes was measured using surface plasmon resonance spectroscopy.

Prenylation is an important modification on peripheral membrane binding proteins. Insect cells can be manipulated to produce farnesylated and carboxymethylated KRAS4b in quantities that enable biophysical measurements of protein-protein and protein-lipid interactions

完全加工重组KRAS4b:分离和描述脱脂和甲基化蛋白

Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human ...