这个项目是由Knut和Alice Wallenberg基金会和瑞典研究理事会资助。

1。 gene/ABDz1-tagged目标融合克隆构建<ol><li>准备合适的酶切位点，在靶基因的表达质粒（质粒含有ABDz1是免费的，通过材料转让协定）的N -末端结扎两侧ABDz1基因的PCR片段。 </li><li>顺劈斩纯化的表达载体，并在一个适当的反应缓冲区选择的限制性内切酶的PCR片段。净化结扎前产品。 </li><li>结扎包含感兴趣的基因的表达载体的限制ABDz1片段，连接产物转化至E.大肠杆菌 （RR1ΔM15应变，在原来的方法是使用 8） 。推广转化辅以选择合适的抗生素的琼脂平板上的细胞。 </li><li> PCR扩增屏幕几个殖民地和序列验证通过DNA测序的结果表达盒。 </li><li>准备质粒序列verif过夜培养IED的殖民地，转化为首选的表达菌株（在原来的方法大肠杆菌罗塞塔（DE3），质粒，以改善人类基因编码的蛋白质的生产托管pRARE，被用来）。 </li></ol> 2。蛋白表达<ol><li>单一菌落接种到5 g / L酵母提取物和适当的抗生素辅以胰蛋白酶大豆肉汤10毫升。在150 RPM孵育37℃过夜。 </li><li>在100毫升的新鲜培养基接种1毫升过夜培养，当细胞达到对数生长期，诱导蛋白表达（异丙基-β- D -半乳糖苷时，lac操纵子是利用原来1毫米）。 </li><li>在150转离心25 ° C过夜和收获细胞孵育。 </li></ol> 3。正交亲和纯化<ol><li>在25毫升的运行缓冲液（的Tris - HCl 25毫米，200毫米氯化钠，1毫米EDTA，0.05％（W / V）吐温20，pH值8.0）重悬沉淀和扰乱次通过超声Ë细胞在60％的幅度1.0/1.0脉冲为3分钟。离心机的样品和筛选靶蛋白，含上清（0.45微米），然后再进一步净化。 </li><li>平衡1毫升血清白蛋白琼脂糖凝胶柱或NHS活化的列，用10个柱体积（CV）的1毫升/分钟运行在一个合适的蛋白质纯化系统的缓冲区的前面加上与HSA根据供应商的建议，。细菌裂解液0.5毫升/分钟的负载，然后复位1毫升/分钟的流速。 </li><li>洗5 5洗涤液的简历（5毫米NH 4 AC，pH值5.5）的运行缓冲液的简历列。 </li><li>洗脱缓冲液1毫升/分钟（0.5米的HAc，pH值2.5）洗脱样品，收集馏分，显示器在280 nm处的吸收，选择分数为进一步净化洗脱峰。 </li><li>池与蛋白浓度最高的分数，当然运行缓冲稀释2倍（20毫米，150毫米氯化钠，pH 7.2磷酸）和确保pH值大约是中性的。新增1个M的Tris - HCl pH值为8，增加pH值，如有必要，。 </li><li> 1毫升HiTrap MabSelect当然列已在1毫升/分钟与10 CV当然运行缓冲平衡，负载在0.5毫升/分钟的样品。重设流速为1毫升/分钟加载后。 </li><li> 5确保运行缓冲的简历（步骤5）洗柱和洗脱的蛋白质与0.2 M醋酸，pH值2.7。对于敏感的目标蛋白质的洗脱液可以瓦解后，收集直接由添加的Tris - HCl。 </li></ol>确定，如果色谱的步骤是颠倒洗脱液的MabSelect列可以运行缓冲液（步骤3.1）稀释，pH值增加了1米的Tris - HCl此外，约8。在一般情况下，窄峰时受雇于在第二个步骤是确保矩阵。 4。十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS - PAGE电泳纯度评价） <ol><li>负载上的纯化组分降低SDS - PAGE和，如果需要，通过质谱分析纯化产物的分子量。重要的是要充分降低的样本，因为在ABDz1免费半胱氨酸可能会导致非还原条件​​下的产品二聚。 </li></ol> 5。代表性的成果: 作为一项原则的证明，正交亲和纯化的ABDz1标签促进三个人类的目标，代表不同的溶解度类，分子量和等电点（见表1）的蛋白质进行了评估。 ABDz1基因基因融合靶基因在大肠杆菌中表达和结构，图2显示了在连续的方法的所有步骤的流程图。经净化正交协议，在不同的点采集的样品进行了分析，通过SDS - PAGE（图3）。结果清楚地表明了双标记和两个非常具体的纯化步骤的效用。即使合理的纯度是AChieved从凝胶的初步净化步骤后，连续步骤产生非常纯净的产品，非常适合要求极高的应用。 <img alt="图1" src="/files/ftp_upload/3370/3370fig1.jpg" /> 图1:用于选择双功能ABDz1标签的组合库设计。 46个氨基酸的白蛋白结合成一个稳定的三螺旋束域的褶皱和它包含了人血清白蛋白的结合位点主要位于第二个螺旋。通过基因随机11个表面暴露在位于第一和第三的螺旋氨基酸，一种新型的具有约束力的表面工程。图中的11个职位表示，和编号Kraulis等。 。以下对金黄色葡萄球菌蛋白的Z域噬菌体表达的组合库，ABDz1的双功能分子被认定二聚体biopanning。 <i毫克ALT ="图2"SRC ="/ files/ftp_upload/3370/3370fig2.jpg"/&gt; 图2正交亲和纯化协议的一个简化的流程图。 一个PCR片段包含ABDz1序列结扎（N端）与有兴趣在一个合适的表达载体的基因。结扎的产品转化为E 。序列验证和质粒制备的大肠杆菌。下面的转换表达宿主，一个大规模的重组蛋白表达的文化是从最初的过夜培养。离心收集细菌，超声裂解产生含有靶蛋白的细菌裂解。经过过滤的步骤，以去除残留的固体颗粒，液体处理系统受到裂解经历连续两个纯化步骤正交亲和纯化。洗脱峰从两个纯化步骤采样，并通过SDS - PAGE进行评估，以评估纯度和比到T他裂解纯化前。 <img alt="图3" src="/files/ftp_upload/3370/3370fig3.jpg" /> 图3。ABDz1在融合表达和纯化目标蛋白质的SDS - PAGE分析。 从融合ABDz1，代表不同的属性，和ABDz1标签本身所表达的三种蛋白的细菌裂解的样品采集，连同相应的山峰上的HSA柱净化样品由MabSelect肯定列（一） 。泳道1-4代表从裂解液净化，5-8车道从HSA的净化（第一步）和9-12的MabSelect肯定净化（第二步）车道前的样品。样品的加载顺序如下:ABDz1 - 141377，ABDz1 HT875，ABDz1 HT2375和ABDz1。此外，从纯化，以相反的顺序相同的列相同的裂解物获得的样本进行了分析（二）。泳道1-3代表从裂解物样品之前，净化，车道4-6 fROM MabSelect肯定净化（第一步）和7-9车道从HSA的净化（第二步）。样品被装在相同的顺序（一），但不包括标签本身。从这些结果很明显，这两个步骤的方法可以得到高度纯化的蛋白质，无论在其中的步骤的应用顺序。 <table border="1"><tbody><tr><td> 名称 B </td><td> 靶蛋白 Uniprot 彗星 </td><td> 分子 重量（KDA） </td><td> 可溶性 D 类 </td><td> 分子量 融合的产物（KDA） </td><td> 等电点 融合的产物 </td></tr><tr><td> 141377 </td><td> B7Z315 </td><td> 17.4 </td><td> 4 </td><td> 23.7 </td><td> 8.5 </td></tr><tr><td> HT875 </td><td> P01040 </td><td> 10.8 </td><td> 3 </td><td> 17.1 </td><td> 4.9 </td></tr><tr><td> HT2375 </td><td> P00740 </td><td> 8.9 </td><td> 5 </td><td> 15.2 </td><td> 6.8 </td></tr></tbody></table><ol class="lalpha"><li> ABDz1标签单独的分子量为6.3 kDa的，等电点为6.7。 </li><li>目标蛋白质表示的Uniprot蛋白质的一部分。 </li><li> <a href="http://www.uniprot.org">http://www.uniprot.org</a> 。 </li><li>溶解度类蛋白N -端片段他的6个总部基地10。 </li></ol>表1。 ABDz1标记靶蛋白与融合产品的一个评估的原则，研究证明。 正交亲和力的方式，选择不同的分子量，溶解度，等电点的三个独特的人体蛋白质的表达和纯化。

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	<li>Waugh, 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=Making+the+most+of+affinity+tags.">Making the most of affinity tags.</a> Trends Biotechnol. 23, 316-320 (2005).</li><li>Hedhammar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+engineering+strategies+for+selective+protein+purification.">Protein engineering strategies for selective protein purification.</a> CET. 28, 1315-1325 (2005).</li><li>Porath, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+chelate+affinity+chromatography,+a+new+approach+to+protein+fractionation.">Metal chelate affinity chromatography, a new approach to protein fractionation.</a> Nature. 258, 206-213 (1975).</li><li>Linhult, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutational+analysis+of+the+interaction+between+albumin-binding+domain+from+streptococcal+protein+G+and+human+serum+albumin.">Mutational analysis of the interaction between albumin-binding domain from streptococcal protein G and human serum albumin.</a> Protein Sci. 11, 206-213 (2002).</li><li>Nilsson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+synthetic+IgG-binding+domain+based+on+staphylococcal+protein.">A synthetic IgG-binding domain based on staphylococcal protein.</a> A. Protein Eng. 1, 107-113 (1987).</li><li>Alm, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small+bispecific+protein+selected+for+orthogonal+affinity+purification.">A small bispecific protein selected for orthogonal affinity purification.</a> Biotechnol. J. 5, 605-617 (2010).</li><li>Nilsson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+affinity+domains+for+the+detection,+purification+and+immobilization+of+recombinant+proteins.">Multiple affinity domains for the detection, purification and immobilization of recombinant proteins.</a> J. Mol. Recognit. 9, 585-594 (1996).</li><li>R&#252;ther, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pUR+250+allows+rapid+chemical+sequencing+of+both+DNA+strands+of+its+inserts.">pUR 250 allows rapid chemical sequencing of both DNA strands of its inserts.</a> Nucleic Acids Res. 10, 5765-5772 (1982).</li><li>Kraulis, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+serum+albumin-binding+domain+of+streptococcal+protein+G+is+a+three-helical+bundle:+a+heteronuclear+NMR+study.">The serum albumin-binding domain of streptococcal protein G is a three-helical bundle: a heteronuclear NMR study.</a> FEBS Lett. 378, 190-194 (1996).</li><li>Hedhammar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+flow+cytometry-based+method+for+analysis+of+expression+levels+in+E+coli+giving+information+about+precipitated+and+soluble+protein.">Novel flow cytometry-based method for analysis of expression levels in E coli giving information about precipitated and soluble protein.</a> J. Biotechnol. 119, 133-146 (2005).</li><li>Carter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+proteolysis+of+fusion+proteins.">Site-specific proteolysis of fusion proteins.</a> ACS Sympos. Ser. 427, 181-193 (1990).</li><li>Hedhammar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negatively+charged+purification+tags+for+selective+anion-exchange+recovery.">Negatively charged purification tags for selective anion-exchange recovery.</a> PEDS. 17, 779-786 (2004).</li><li>Gr&#228;slund, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charge+engineering+of+a+protein+domain+to+allow+efficient+ion-exchange+recovery.">Charge engineering of a protein domain to allow efficient ion-exchange recovery.</a> Protein Eng. 13, 703-709 (2000).</li></ol>

没有利益冲突的声明。

这里介绍的正交亲和纯化的协议，使广泛的靶蛋白的高效净化。通过与白蛋白结合域新的结合表面的结合位点相结合的内在，开发一个小的双功能蛋白标记。它是非常简单的使用标签，因为它可以简单地克隆和融合表达任何蛋白质的首选任何表达载体的兴趣。在大多数实验室提供的标准设备，可受聘为两个步骤的蛋白质纯化。由于ABDz1标签的功能取决于有效地揭露其两个结合面域的正确折叠，方法是有限的可溶形式表达的蛋白质和不适合在变性条件下纯化。还原剂也应避免，因为它们干扰ABDz1约束力MabSelect当然矩阵z域。 正交亲和纯化TION提供了一个有用的替代传统的方法，以满足高度纯化的蛋白质在许多苛刻的应用要求。同时，这种方法不再需要时连续使用不同的净化策略组合标记。对于需要一个本地的靶蛋白的应用程序，一种蛋白酶裂解位点可能出台使酶去除ABDz1标签可能11。此外，ABDz1标签所述的第一双特异性小而折叠的蛋白质结构域。它是独一无二的，因为它提供了一个净化战略，取决于两个目标的具体亲和力相互作用。在未来，这将是有趣扩大这个概念，开发新的双功能标签，进行与现成的和廉价的色谱树脂兼容的新的具有约束力的表面。例如，通过参与白蛋白结合的氨基酸取代基本的残留物，一个标签与I兼容交换层析可能实现的。类似的概念已被证明有效的Z域由负责工程，分别生成已知标记为Z 酸 12和Z 基本 13兼容阴离子和阳离子交换。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> E.大肠杆菌 RR1ΔM15 </td><td>美国典型培养物保藏</td><td> 35102 </td></tr><tr><td> E.大肠杆菌罗塞塔（DE3） </td><td> Novagen公司</td><td> 70954 </td></tr><tr><td>胰蛋白酶大豆肉汤</td><td> DIFCO </td><td> 211822 </td></tr><tr><td>酵母提取物</td><td> DIFCO </td><td> 212720 </td></tr><tr><td>振动细胞sonicator </td><td>超音速队和材料</td><td> - </td></tr><tr><td> HSA的琼脂糖凝胶</td><td>法玛西亚生物技术</td><td>前产品</td></tr><tr><td> HiTrap MabSelect当然</td><td> GE医疗集团</td><td> 11-0034-93 </td></tr><tr><td> HiTrap NHS活化HP列</td><td> GE医疗集团</td><td> 17-0716-01 </td></tr></tbody></table>

orthogonal protein purification facilitated by a small bispecific affinity tag

由于相关的协议需要合理化的重组蛋白的纯化成本高。对于高吞吐量的努力，是一个一般的方法不需要目标蛋白质的特定优化1的需求。要做到这一点，纯化，基因可以融合到感兴趣的基因标签是常用的2。使用最广泛的亲和力处理的六溴氨酸标签，这是进行净化，适合在本地和变性条件3。生产标签的代谢负担低，但它不提供高特异性的亲和层析1,2为基础的战略。 在这里，有两种不同的结合位点上的46个氨基酸的双特异性纯化标签，小的蛋白质结构域已被开发。白蛋白结合域是来自链球菌G蛋白，人血清白蛋白具有很强的固有的亲和力（HAS）。 11个表面暴露的氨基酸，不涉及白蛋白结合4，基因随机产生一个组合库。有人对噬菌体颗粒随机安排结合表面（图1）小说中的蛋白质库噬菌体展示技术，以方便选择粘合剂。经过几轮针对二聚体biopanning z域从金黄色葡萄球菌蛋白A 5，小双功能分子，具有亲和力HSA和新颖的目标是确定的6派生。 新的蛋白质结构域，简称为ABDz1，被评为为选择目标蛋白质具有不同的分子量，溶解度，等电点的纯化标签。三个目标蛋白的N -末端融合，此后亲和纯化的小说标签表示在Escherishia大肠杆菌 。初步净化与固定的HSA或z域的列导致relatively纯的产品。两步双特异标记的亲和纯化，蛋白纯度大幅改善。色谱媒体与Z域的固定化，例如MabSelect当然，是现成的抗体纯化和HSA的化学可以很容易地被加上媒体提供的第二个矩阵。 这种方法尤其有利时，有一个恢复的目标蛋白的纯度高的需求。要使用两种不同的层析步骤bifunctionality标签允许表达宿主的代谢负担是有限的，由于体积小标签。所谓组合标记多个标签1,7组合使用，它提供了一个竞争力的替代。

Watch this Scientific Journal Video about Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag at JoVE.com

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

已开发一种新的和高效两步亲和层析协议，并进行了详细描述。该方法是基于一个小净化标签有两个固有的亲和力，适用于广泛的目标蛋白质具有不同的属性。

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 ...

orthogonal-protein-purification-facilitated-small-bispecific-affinity

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17.0K 次观看.  Royal Institute of Technology. 已开发一种新的和高效两步亲和层析协议，并进行了详细描述。该方法是基于一个小净化标签有两个固有的亲和力，适用于广泛的目标蛋白质具有不同的属性。

视频: Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

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

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

由不稳定的多蛋白质复合物的分离在体内控制细胞的交联和免疫磁性亲和层析

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

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 ...

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 ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

蛋白复合物的鉴定<em&gt;大肠杆菌</em使用顺序肽亲和纯化技术相结合串联质谱法

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

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

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

高通量的亲和标签的重组蛋白的纯化

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

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness1. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)2,3 and a C-terminal 10xHis tag4, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression5. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme6. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.

In the present protocol, we demonstrate a highly efficient and cost-effective small-scale protein purification method, which allows purification of recombinant proteins by uniquely combining a cleavable GST-tag and a small His-tag.

GST-他的净化:一个两步亲和纯化协议屈服全长纯化的蛋白质

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. ...

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 ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

一个协议，用于噬菌体展示和亲和选择以重组蛋白诱饵

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

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. ...

由一个小型的双特异性亲和标签的推动正交蛋白质纯化

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