这项工作是支持由来自美国国立卫生研究院（5T32GM008275 22）和美国心脏协会predoctoral的奖学金（EAS）的赠款;化学生物学界面从美国国立卫生研究院（2T32GM071339 06A1）（MED）奖学金;和赠款，从国立卫生研究院（1DP2OD002177 - 01和NS067354 - 0110），埃利森医学基金会，比尔和梅林达盖茨基金会（JS）。

1。对Hsp104的表达<ol><li>质粒在大肠杆菌中聘用为净化大肠杆菌 ，pPROEX HTB - Hsp104，包含的trc启动子的诱导 26控制下Hsp104开放阅读框。质粒产生的N -端他的6个标签，可删除TEV蛋白酶裂解Hsp104。转换成密码子优化E. pPROEX HTB - Hsp104 大肠杆菌 BL21 CodonPlus的RIL细胞（Stratagene公司，安捷伦科技公司）使用一个典型的细菌转化过程（例如按照制造商的指示）。重要的是要使用一个密码子优化的E.菌株 ，因为Hsp104有一个不寻常的密码子的偏见。 </li><li>接种（USB.方药：16克/ L，酪蛋白胨，10G / L酵母提取物，5克/ L的NaCl，pH值7.0），辅以新鲜转化100μg/mL氨苄青霉素和34μg/mL氯霉素一个100毫升2XYT文化和成长一夜之间在37 ° C在200rpm摇晃。 </li><li>过夜培养，接种到6 X 1L100μg/mL氨苄青霉素和34μg/mL氯霉素2XYT在2L烧瓶10ML。在250rpm摇在37 ° C，直到OD 600 = 0.4-0.6。停止震动和降低温度至15 ° C。允许细胞以平衡为30min unagitated在孵化器，直到它到达15 ° C。一旦15 ° C时达到诱导蛋白的表达，加入IPTG至终浓度为1mm。继续晃动在250rpm过夜（12-16小时）。 </li></ol> 2。细胞收获和裂解<ol><li>为20分钟4000转离心收集细胞（在预冷转子）在4 ° C（我们使用一个Sorvall RC 3BP +离心机）。后续步骤必须立即执行，因为Hsp104活动减少时，大肠杆菌大肠杆菌细胞被冻结。此外，所有的后续步骤，应在冰上或4℃ </li><li>在prechilled悬浮细胞球（冰）10毫升裂解液（40MM的HEPES - KOH pH值7.4，500mm的氯化钾，氯化镁20MM 2，2.5％（W / V）甘油，20MM咪唑，5μM胃酶抑素A，完整的蛋白酶抑制剂的鸡尾酒（1 EDTA的免费tablet/50mL），和2mm的β-巯基乙醇）。 </li><li>一位法国记者（Emulsiflex）使用热交换器，浸泡在冰水中匀浆，裂解细胞。在使用之前，确保所有细胞团块已溶解。平衡与裂解缓冲液匀浆后，通过15,000-18,000 psi的压力缸的传递是足够充分溶解。如果一个法国媒体不可用，通过超声与溶菌酶的潜伏期可以使用（见讨论）。保存一个小样本的裂解细胞，SDS - PAGE分析（1μL）（图2中的裂解液）。 </li><li>细胞碎片取出离心20分钟16,000转，4 ° C（我们使用一个Sorvall RC5C + centifuge）。保留上清液为下一步并保存为一个小样本的上清液（1μL）SDS - PAGE分析（镍负载图2）。 </li></ol> 3。 Hsp104净化<ol><li>从步骤2.4混合上清液12mL（2ML镍琼脂糖珠，每细胞1L）50％的裂解液平衡的镍琼脂糖凝胶珠（GE）的泥浆。 </li><li> 3个小时的样品慢慢旋转4 ° C，镍琼脂糖凝胶均匀悬浮发泡最小化。更短的孵育时间是可能的，但会减少产量。在4 ° C在蛋白酶抑制剂的存在，小的退化发生，和3个小时的镍琼脂糖凝胶孵化时间不会降低最终产品的活动。收集镍琼脂糖离心2分钟，在4 °在2000彗星RPM（的Eppendorf 5810R离心机）。保存一个小样本上清进行SDS - PAGE分析（1μL），并丢弃其余的（镍流经（FT）在图2）。 </li><li>洗洗涤缓冲液（40MM HEPES - KOH pH值7.4，150mm的氯化钾，氯化镁2 20MM，2.5％（W / V）甘油，20MM咪唑，2mm的β-巯基乙醇），5倍柱体积的25倍柱体积的提取镍琼脂糖凝胶用1M KCl的缓冲，以消除静电绑定到Hsp104的污染物，25柱体积的缓冲液洗。镍琼脂糖收集每个离心洗2分钟后，4 °在2000彗星RPM（的Eppendorf 5810R离心机）。每个洗，离心循环后，吸删除缓冲区。 </li><li>洗脱1ML洗脱缓冲液制成每1ml镍琼脂糖（40MM HEPES - KOH pH值7.4，150mm的氯化钾，氯化镁2 20MM，2.5％（W / V）甘油，350MM咪唑，2mm的β-巯基乙醇）和Hsp104，混合镍琼脂糖凝胶在4 ° C 20分钟旋转。咪唑浓度高破坏的倪珠他6互动。镍琼脂糖凝胶取出空的1.2毫升离心柱（BIO - RAD）离心2分钟4 °在2,000 RPM（的Eppendorf 5810R离心机）彗星。保存洗脱液SDS - PAGE分析1μL（倪洗脱液图2）。 〜15毫克的Hsp104是每公升的细胞，在这一步获得。 </li><li>采用截留分子量为30,000 Amicon超（MIL缓冲区交换到缓冲区问（20MM的Tris - HCl pH值8，0.5mm的EDTA，MgCl 2的 5MM，50MM氯化钠）洗脱液lipore）15ML离心选矿单位。首先，集中〜1.5ml的蛋白质，再加入14.5mL缓冲Q来稀释10倍的蛋白质。缓冲交换，重复3次。增加的pH值和低盐，确保Hsp104具有较高的负电荷。 </li><li>净化Hsp104通过阴离子交换色谱法，使用一个缓冲Q平衡资源Q 6ML列（GE）。注射前，蛋白质进行筛选，通过低蛋白具有约束力的Millex GP PES膜0.22μm的注射器过滤器（Millipore公司）。我们聘请的为1ml/min的流速。洗去弱结合蛋白1柱体积的20％，缓冲区Q +（20毫米的Tris - HCl pH值8，EDTA的0.5MM，5MM氯化镁2，1M氯化钠）。洗脱Hsp104超过5倍柱体积与线性渐变（20％-50％Q +缓冲）（图3）。收集1毫升分数。 Hsp104和大多数变种通常〜400mm的氯化钠（31mS/cm）洗脱。保存样品的SDS - PAGE分析（图3，插图）的最高分数为（1μL）。 </li><li>为了消除他的6个标签，使用Amicon离心选矿机的交换蛋白上面描述（见第3.5步）进入裂解缓冲液（20MM的HEPES - KOH pH值7.4，140mm的氯化钾，和10mm氯化镁2 ）。根据制造商的说明使用proTEV蛋白酶（Promega公司）或AcTEV蛋白酶（Invitrogen公司）。 Hsp104，我们发现，比例较高的TEV蛋白酶：Hsp104充分裂解。我们使用了1％12μgHsp104蛋白酶单位的比例。应在30 ° C卵裂为2-4小时，16小时的潜伏期在4 ° C。最后Hsp104浓度应介于20 -75μm的单体。加入过量的镍琼脂糖凝胶的Hsp104在裂解反应量（假设珠可以绑定15毫克每毫升包装树脂的蛋白质）消耗任何His6标签的​​剩余Hsp104镍琼脂糖凝胶（GE）和TEV蛋白酶。删除珠空离心柱（BIO - RAD）在2分钟2,000转离心4 ° C。 SDS - PAGE分析裂解（1μL）之前和之后收集的样本，以确保乳沟，uncleaved蛋白质的去除（图4）。 </li><li>兑换成体积排阻采用截留分子量为30,000 Amicon超（Millipore公司），在步骤3.5中所述的缓冲区（20MM的HEPES - KOH pH值7.4，氯化钾140MM，10MM氯化镁2，EDTA的0.1MM，1MM数码地面电视）。 </li><li>进一步净化Hsp104通过体积排阻色谱。使用体积排阻缓冲平衡Superose 6或Superdex 200柱（均来自GE）（图5）。注射前，蛋白质进行筛选，通过低蛋白具有约束力的Millex GP PES膜0.22μm的注射器过滤器（Millipore公司）。 10/300大小列（24mL）小于10毫克的蛋白质，可用于0.5毫升馏分收集。 12/60大小列（120ML）超过10毫克的样品，应使用1毫升馏分收集。请参阅制造商的流动速率和样品装入卷指示。经纯化后，Hsp104分数汇总，如图所示。 5和集中Amicon离心浓缩装置（Millipore公司）。 Hsp104存储，如下所述。由于分离Hsp104降解产物和污染物的最终产量的纯化全长Hsp104〜1 - 3mg的每开始文化公升的物质损失。 </li></ol> 4。 Hsp104 Disaggregase活动<ol><li>净化后，它是很好的做法，在分类检测评估Hsp104活动。通常情况下，我们采用荧光素酶的激活检测（图6） 。在这个实验中，结合与HSP70的伴侣系统（Hsp70和Hsp40）分解尿素变性的萤火虫荧光素酶聚合2 Hsp104。可溶性的荧光素酶催化氧化荧光素oxyluciferin，反应，释放光。通过监测发光，相对激活，因此分类，荧光素酶才能确定。 Hsp104必须能够协同合作与HSP70的合作伴侣系统。恢复发光量取决于确切的热休克蛋白70：Hsp40对利用。我们经常聘请HSP72和Hsp40（分析设计）。至少，积极Hsp104应该产生在荧光素酶的激活增加5倍，相比HSP72激活：Hsp40（图6）。 Hsp104的筹备工作，没有达到这个水平的活动都将被丢弃。 </li></ol> 5。 Hsp104存储<ol><li>短期储存，Hsp104可以保持在4 ° C体积排阻缓冲区。然而，活动将下降2-3天之后，在4 ° C和1周后大幅4 ° C。对于分类分析，我们建议，Hsp104应采用净化后尽快。理想的情况下，Hsp104应立即使用淀粉样蛋白分解实验。 </li><li>如果蛋白质必须长期存储，Hsp104交换到存储缓冲区（20MM的HEPES - KOH pH值7.4，氯化钾140MM，10MM氯化镁2，EDTA的0.1MM，1MM数码地面电视，10％甘油（W / V））。 100μL等分是液氮冷冻和储存在-80 ° C的管理单元冻融循环次数大大减少Hsp104活动应尽量避免。我们建议慢慢解冻冰Hsp104。 -淀粉样蛋白disaggregase的活性会大幅下降1个月后，在-80 ° C。 </li></ol> 6。代表性的成果和数据： <img src="/files/ftp_upload/3190/3190fig1.jpg" alt="图1"/> 图1。 Hsp104是一个双功能的disaggregase。无序聚集（左所示）进行分类，要求合作的HSP70的伴侣系统（Hsp70和Hsp40）2。 Hsp104重塑下令没有援助，Hsp70和Hsp40 在体外淀粉样蛋白聚合体（如右图所示），但Hsp70和Hsp40可以提高对淀粉样蛋白26,28 Hsp104活动。对于这两种类型的聚合结构，Hsp104夫妇ATP水解底物，通过其中央通道，以促进分类易位。从事酪氨酸轴承孔循环，并通过中央通道穿梭基板49-52。 <img src="/files/ftp_upload/3190/3190fig2.jpg" alt="图2"/> 图2。镍亲和纯化步骤的SDS - PAGE电泳分析 。裂解液，镍，镍负载FT和镍洗脱液样品使用4-20％的Tris - HCl1.0毫米标准胶（BIO - RAD）和考马斯染色的SDS - PAGE分割。请注意，并非所有的Hsp104是能够绑定到镍琼脂糖。广泛分子量标记（BIO - RAD）（左线）。 <img src="/files/ftp_upload/3190/3190fig3.jpg" alt="图3"/> 图3。资源Q净化镍琼脂糖洗脱液在280nm处和绿线，蓝线代表的吸光度代表％缓冲区Q +（最高50％ ）。的第一个高峰，其中20％洗脱Q +含有杂质，降解产物和不当折叠Hsp104的。第二和主要峰包含正确折叠和活跃Hsp104。流速为1ml/min。从20-50％Q +梯度是30分钟或5倍柱体积。插图：峰组分经SDS - PAGE分析使用4-20％的Tris - HCl1.0毫米标准胶（BIO - RAD）和考马斯染色解决。广泛分子量标记（BIO - RAD）（左）所示。 <img src="/files/ftp_upload/3190/3190fig4.jpg" alt="图4"/> 图4。 proTEV蛋白酶裂解步骤SDS - PAGE分析 ，从资源Q净化他的6 Hsp104治疗proTEV 4小时蛋白酶在30℃，然后16个小时，在4 ° C。样品经SDS - PAGE分次使用的4-20％的Tris - HCl1.0毫米标准胶（BIO - RAD）和考马斯染色。请注意，proTEV裂解Hsp104迁移更迅速。 TEV蛋白酶和uncleaved Hsp104已经所剩无几步骤3.7中所述的镍琼脂糖凝胶。广泛分子量标记（BIO - RAD）（左）所示。 <img src="/files/ftp_upload/3190/3190fig5.jpg" alt="图5"/> 图5。体积排阻Hsp104净化。劈开Hsp104是通过Superose 6凝胶过滤柱（10/300，GE）进一步纯化。流量= 0.4ml/min。峰之间的虚线代表汇集分数。插图：峰组分经SDS - PAGE分析使用4-20％的Tris - HCl1.0毫米标准胶（BIO - RAD）和考马斯染色解决。广泛分子量标记（BIO - RAD）（左）所示。 <img src="/files/ftp_upload/3190/3190fig6.jpg" alt="图6"/> 图6。荧光素酶的激活检测 。变性萤火虫荧光素酶聚合（50纳米）与HSP72和Hsp40（1μM）（含量设计），Hsp104（6μM单体）或Hsp104，HSP72和90分钟Hsp40在25 ° C孵育变性荧光素酶只有充分激活Hsp104和Hsp40/Hsp72存在。恢复发光，是衡量一个无限M1000酶标仪（TECAN）。价值观代表表示± SEM（N = 3）。

没有利益冲突的声明。

时间轴：最大的Hsp104活动中，我们建议尽可能迅速完成整个净化计 ​​划。然而，纯化步骤，使一个苛刻的时间表，可能并不总是实际的。如果净化步骤是尽快地进行，时间从隔夜表达年底通过孵化2-4小时30 ° C是与TEV蛋白酶约9-11小时。一个潜在的地方，暂停以下TEV裂解步骤。如果绝对必要的，Hsp104可能会被冻结这一步后，上文所述（步骤5.2）的管理单元。解冻后和纯化Hsp104然后必须立即?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td>感受态细胞BL21 - CodonPlus - RIL </td><td> Stratagene公司，安捷伦科技公司</td><td> 230255 </td></tr><tr><td> 2XYT肉汤</td><td> USB </td><td> 75864 </td></tr><tr><td>完成后，迷你型，EDTA无蛋白酶抑制剂药片</td><td>罗氏公司</td><td> 1836170 </td></tr><tr><td>胃酶抑素一个</td><td>西格玛</td><td> P4265 </td></tr><tr><td>镍琼脂糖凝胶6快流</td><td> GE医疗集团</td><td> 17-5318-02 </td></tr><tr><td> Amicon超15离心式过滤器（截留分子量30,000） </td><td> Millipore公司</td><td> UFC903008 </td></tr><tr><td>资源Q - 6毫升列</td><td> GE医疗集团</td><td> 17-1179-01 </td></tr><tr><td> proTEV蛋白酶</td><td> Promega公司</td><td> V6052 </td></tr><tr><td> AcTEV蛋白酶</td><td> Invitrogen公司</td><td> 12575015 </td></tr><tr><td> Superose 6 10/300 GL </td><td> GE医疗集团</td><td> 17-5172-01 </td></tr><tr><td> Hsp40 </td><td>检测设计</td><td> SPP - 400 </td></tr><tr><td> HSP72 </td><td>检测设计</td><td> ADI公司的NSP - 555 </td></tr></tbody></table>

purification of hsp104, a protein disaggregase

Hsp104是一个六聚AAA +蛋白1，从酵母，这夫妇ATP水解蛋白质分解2-10（图1）。这个活动赋予了两个关键的选择性优势。首先，由Hsp104无序聚集复性酵母生存的授权后，各种蛋白质的错误折叠强调，包括热休克3,5,11,12。其次，Hsp104交叉β-淀粉样纤维的重塑使酵母利用无数的朊病 ​​毒（感染性淀粉样）和水库作为一个有益的遗传表型变异13-22。值得注意的是，Hsp104直接重塑preamyloid低聚物和淀粉样纤维，包括酵母朊蛋白Sup35 和 Ure2 23-30组成的。这淀粉样蛋白重塑的功能是专业方面的酵母Hsp104。的大肠杆菌大肠杆菌同源，ClpB，无法改造preamyloid低聚物或淀粉样纤维26,31,32。 Hsp104同源除外，令人困惑，动物王国的生活。事实上，无论是动物细胞中拥有任何酶系统，夫妻蛋白复性（而不是退化）的分类仍然是未知 33-35 。因此，我们和其他人提出，Hsp104可能会被视为一个与特定的蛋白质错误折叠成有毒的preamyloid低聚物和淀粉样纤维 4,7,23,36-38有关的各种神经退行性疾病的治疗剂开发。有没有直接目标与这些疾病有关的汇总物种的治疗。然而，Hsp104溶解毒性低聚物和淀粉样纤维的α-突触核蛋白，这是帕金森氏病23以及39的PrP淀粉样蛋白形式相连组成。更重要的是，Hsp104减少蛋白质聚集和改善帕金森氏病23和亨廷顿氏病38的老鼠模型的神经退行性疾病。理想的情况下，优化治疗和减少副作用，Hsp104将设计和potentiated有选择性地改造的核心问题4,7的疾病的具体聚集。然而，有限的结构和机理的了解如何Hsp104分解聚合结构和不相关的蛋白质的这样一个多样化的剧目挫败这些努力30,40-42。 要了解Hsp104的结构和机制，它是用最少的元件必须研究的纯蛋白质和重组其disaggregase活动。 Hsp104是一个102kDa蛋白与PI〜5.3，ADP或ATP，或在高蛋白质浓度在43-46核苷酸的情况下hexamerizes。在这里，我们描述了一个优化的协议， 从 E的纯化高度活跃，稳定Hsp104 大肠杆菌 。 E.使用大肠杆菌可以简化大规模生产，我们的方法可以进行快速，可靠地为众多Hsp104变种。我们的协议，增加Hsp104的纯度和简化他的6个标签去除，相比以前的提纯方法从E. 大肠杆菌 47。此外，我们的协议是两个较近期的协议 26,48比更轻便和方便。

Watch this Scientific Journal Video about Purification of Hsp104, a Protein Disaggregase at JoVE.com

Purification of Hsp104, a Protein Disaggregase

在这里，我们描述一个高度活跃Hsp104，一个六聚AAA +从酵母中的蛋白质，其中夫妇ATP水解蛋白分解净化的协议。该计划利用从亲和纯化His6标签的​​构造<em&gt; E。大肠杆菌</em&gt;其次，His6标签去除TEV蛋白酶，体积排阻色谱阴离子交换层析。

Hsp104纯化，蛋白质Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

purification-of-hsp104-a-protein-disaggregase

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JoVE Journal

Biology

17.3K 次观看.  University of Pennsylvania. 在这里，我们描述一个高度活跃Hsp104，一个六聚AAA +从酵母中的蛋白质，其中夫妇ATP水解蛋白分解净化的协议。该计划利用从亲和纯化His6标签的​​构造 E。大肠杆菌其次，His6标签去除TEV蛋白酶，体积排阻色谱阴离子交换层析。

视频: Purification of Hsp104, a Protein Disaggregase

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

科研

生物学

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

纯化 M。 magneticum应变AMB - 1磁小体相关蛋白MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP) 1-8. The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC) 9-11. In this example, we use an RND protein, the P. aeruginosa MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.

In this protocol we demonstrate the expression, solubilization, and purification of a recombinantly expressed membrane protein, MexB, as a soluble protein detergent complex. MexB is a multidrug resistance membrane transporter from the opportunistic bacterial pathogen Pseudomonas aeruginosa.

表达，洗涤剂溶解，和一个膜转运，MexB多药耐药蛋白的纯化

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

自动疏水层析蛋白质纯化用柱选择

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

高活性酵母核糖体的色谱分离纯化

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

由一个小型的双特异性亲和标签的推动正交蛋白质纯化

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

鉴定蛋白质相互作用的合作伙伴使用串联亲和纯化

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

侵入细胞DISC1蛋白种类的产生，纯化及表征

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

蛋白质纯化方法测定结合亲和力微尺度热泳

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

鉴定蛋白质 - 蛋白质相互作用的<em&gt;果蝇</em&gt;成人头由串联亲和纯化（TAP）

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...