这项研究是由威康信托高级奖学金颁发给伊恩·古德费洛博士。

1。代细胞系：pMEP4转/表达<ol><li>与pMEP4表达载体转染细胞和潮霉素B /毫升（罗氏）与100微克的选择，直到所有的模拟转染细胞被杀害。在pMEP4质粒保持episomally所以没有必要隔离特定的克隆。 </li><li> 10μM2 CDCL 16小时治疗，可诱发细胞包含pMEP4向量。应证实前小规模扩增的细胞株的表达和诱发。作为G蛋白域几乎所有种类的抗体结合，可以很容易地检测到的TAP标签蛋白。对于大规模纯化通常为10合流175厘米2的细胞瓶是必需的。这相当于约2 X10的8表 ​​达细胞。 </li></ol> 2。细胞裂解液的准备<ol><li>刮至PBS的细胞，并结合成一个单一的50毫升管。旋转1200X GF或5分钟（这应该导致在2-3毫升包装细胞开始，清洗后减少到约150毫升）。 </li><li>在冰冷的PBS（每次50毫升）洗细胞三次。 </li><li>在5毫升裂解液（裂解细胞50毫米的Tris-HCl（pH值7.5），125 mM氯化钠，5％甘油，0.2％的NP-40，MgCl 2的 1.5毫米，25毫米，1毫米氟化钠钠4 3旁白和蛋白酶抑制剂）。注：氟化钠，钠3 VO 4和蛋白酶抑制剂应补充新鲜。吸取了前5分钟在冰上留下的10倍。离开冰再次在冰上5分钟前使用钝针注射器上升和下降的5-10倍。重复syringing和离开进一步冰上5分钟。 </li><li>冻融裂解两次（液体N 2 /或干冰块和乙醇）。不要让样品达到大于4°C。注意：您可以储存在-80°C，直到你有时间来处理这个范例。 </li><li>分装到1.5毫升管样本和删除unlysed细胞和Debris通过离心（在4°C，16,000 XĞ10分钟）。 </li><li>恢复上清液，在15毫升猎鹰管相结合，并通过0.45微米的过滤（可选）前通过采取定量蛋白质产量的20μL样品。卸下进一步的后续分析（样品1）50μL等份。 </li></ol> 3。结合兔IgG-琼脂糖 （注：应进行所有的自旋1200XĞ在冷冻离心机1分钟，在4°C，除非另有说明） <ol><li>纷飞瓶轻轻悬浮兔IgG-琼脂糖溶液（Sigma-Aldrich公司）。取380μL的IgG琼脂糖（截止1毫升枪头）和离心1分钟，除去航运/防腐剂的解决方案。琼脂糖三次洗冷冻裂解液（4℃）使用离心澄清。琼脂糖的最终产量应该装珠约250μL。 </li><li>从2.6（15毫升猎鹰浴缸中加入清除的细胞裂解液E）水洗兔IgG琼脂糖孵育3小时（或隔夜）在4°C使用一个旋转的搅拌机。 </li><li>降速琼脂糖珠在4°C 5分钟，取出供后续分析（样品2）上清。注：标签蛋白的TAP现在应与珠。 </li></ol> 4。 TEV蛋白酶切割 （注：应进行所有的自旋1200XĞ在冷冻离心机在4℃1分钟，除非另有说明） <ol><li>冷冻裂解液洗兔IgG琼脂糖珠三次（4°C）使用离心澄清（注：裂解液中不应该包含蛋白酶抑制剂）。应小心，不要删除或失去任何珠在洗涤步骤。 </li><li>洗珠TEV蛋白酶裂解缓冲使用离心澄清（10毫米的Tris-HCl（pH值7.5），100 mM氯化钠，0.2％NP-40）进一步两次。最后一次洗涤后小心取出所有李从珠嚼食。 </li><li>准备一个TEV蛋白酶切割组合;每个样品包括467.5μL 氢澳，25μL20X，TEV缓冲（Invitrogen公司），5微升0.1M数码地面电视和2.5μL（25美）TEV蛋白酶（Invitrogen公司）。加入500μl这个TEV酶切组合的每个样品包装珠和整个组合转移到1.7毫升预润滑管（COSTAR）。之前，以孵化为后续分析（样品3）删除30μL等份。 </li><li> TEV蛋白酶反应过夜孵育4℃用一个旋转的搅拌机。请注意，可用于较短的孵化时间取决于诱饵蛋白和无障碍TEV蛋白酶裂解位点，但最小的孵化时间的性质，应凭经验确定。 </li></ol> 5。结合ULTRALINK固定的链霉加珠 （注：应进行所有的自旋1200XĞ在冷冻离心机1分钟，在4°C，除非另有说明） </P&gt; <ol><li>离心TEV酶切反应，其中包含为1200X克（4℃）5分钟的兔IgG琼脂糖珠。删除的分析澄清上清20μL等份（样品4） </li><li>其余上清液（约480μL）转移到一个新的1.5毫升预润滑管（COSTAR）离开冰。不要扔掉此上清 - 这包含TEV切割诱饵蛋白和任何关联的结合蛋白。 </li><li>新增500个冷藏裂解缓冲液（2.3节），其余兔IgG琼脂糖珠和重悬。这种混合物离心澄清之前，去除上清相结合，从步骤5.2珠。离开冰管（它现在应该包含〜980μL的样品）。 </li><li>保留供后续分析兔IgG琼脂糖珠（样品5）。注：如果你运行这个样本上的SDS-PAGE胶（或印迹），它会给很多背景，由于存在的IgG重型和轻型链。 </li><li>同时轻轻悬浮ULTRALINK固定纷飞的瓶子，加链霉亲和素琼脂糖珠（皮尔斯）。使用切成末，预润滑1.5毫升管分装成70μL链亲和素珠每样本200μL枪头和离心除去运输/防腐剂的解决方案。注意：这些珠子都非常小，所以“鸭子嘴”或平/窄端的技巧可以使用，以尽量减少损失洗涤过程中的珠。 </li><li>洗链亲和素珠冰鲜裂解液3次（4°C）使用离心澄清。这应该留下约45-50微升，每管装珠。 </li><li>转让TEV蛋白酶裂解上清（即从步骤5.3样品980μL）管含3小时（或隔夜）洗链亲和素磁珠孵育4℃用一个旋转的搅拌机。 </li><li>降速在4°C 5分钟的链亲和素珠，去除上清，为后续的分析（山姆PLE 6）。洗链亲和素珠冰鲜裂解液3次（4°C）使用离心澄清。最后一次洗涤后，小心地取出所有剩余的洗涤缓冲液。 </li></ol> 6。素的链亲和素的结合肽和诱饵蛋白的洗脱 （注：应进行所有的自旋1200XĞ在冷冻离心机1分钟，在4°C，除非另有说明） <ol><li>加入500μL的PBS洗脱标记的链亲和素珠蛋白生物素（1毫米D-生物素），并在4°C孵育3小时，用一个旋转的搅拌机（或过夜）。 </li><li>降速5分钟，在4°C的链亲和素珠，并取出上清液1.5毫升预润滑管（注：这是你最后的样品/洗脱（7）样品。 </li><li>另外500μL生物素：PBS加入剩余的链亲和素珠，并在4°C孵育2-3小时，用一个旋转的搅拌机（或过夜）。 </li><li>重新泥炭加强6.2，并结合两个洗脱（样本7）。这是最后的洗脱可以储存在-80°C </li><li>为了评估生物素洗脱效率，冻结余下的链亲和素珠，煮沸后在SDS样品缓冲液（样品8）（建议：减少从费舍尔LaneMarker 5倍样品缓冲液）。 </li></ol> 7。蛋白质浓度<ol><li>最后洗脱液（样品）需要分析前要集中，因为低蛋白浓度相对高容量。这可以使用低分子量（&lt;5 KDA）Vivaspin自旋列（Vivascience）。旨在集中量小于100μL。这最后的样本，可煮，蛋白上样缓冲液中（建议：LaneMarker 5倍减少从费舍尔的样品缓冲液）。 </li></ol> 8。分析<ol><li>样品1-8可以通过免疫印迹分析，以确定下拉效率。注意：大多数中学抗体与G蛋白域的融合蛋白发生交叉反应，但这是TEV蛋白酶裂解后删除。 </li><li>可以1D或2D SDS-PAGE电泳分析样品7。可以使用银或考马斯（建议：SilverQuest银染色和蓝色胶体考马斯染色试剂盒自Invitrogen）进行染色。可以用质谱进行蛋白质鉴定。 </li></ol> 9。代表结果 1D的SDS-PAGE分析，从该协议的最终洗脱（样本7）确定的TAP标签eIF4E的结合伙伴的一个例子是在图2。这代表凝胶反映的复杂和丰富的自然与其他蛋白质在细胞中eIF4E的相互作用。在图2所示的阴性对照，从一个细胞只表达TAP标签生成的比较，说明这eIF4E的诱饵下拉的特殊性。在这种情况下集中的最终洗脱15％（样本7）使用Invitrogen公司从Silverquest银染色试剂盒染色前1D使用市售的预制梯度凝胶的SDS-PAGE分析。 通常50-85％，其余集中的最终洗脱（样本7）进行了分析与胶体考马斯染色（Invitrogen公司）。从整个车道的样品（凝胶片/条），然后提取和分析，质谱分析。 eIF4E的下拉鉴定的蛋白质进行过滤对从阴性对照，以确定非特异性结合合作伙伴的具有约束力的合作伙伴。确定最终的蛋白质使用这个TAP标签eIF4E的过程可以看出，在图2中，这是正常的蛋白质确定使用这种技术的代表。此外，还确定了一些eIF3亚基（数据未显示）。 <img src="/files/ftp_upload/3643/3643fig1.jpg" alt="图1" /> 图1。示意图串联亲和纯化过程。六步串联亲和纯化（TAP），协议涉及细胞的生成，细胞裂解，兔IgG琼脂糖凝胶免疫沉淀，TEV蛋白酶裂解，链霉亲和素磁珠亲和纯化，并终于素洗脱。 <img src="/files/ftp_upload/3643/3643fig2.jpg" alt="图2" /> 图2。串联亲和纯化的小鼠eIF4E的蛋白质。互动合作伙伴的N末端 ​​抽头标签eIF4E的使用所附的协议，从真核细胞转染HEK293细胞进行纯化。一个20％的分数，最后洗脱（样本7），通过SDS-PAGE分析，预铸的4-12％梯度凝胶电泳的TAP-eIF4E的车道上。一个单独的标签（TAP车道）进行等效分析。用银染蛋白。随后通过对同一样品质量法确定的蛋白质强调这种凝胶。 缩写：eIF4G;真核翻译起始f演员4伽马，PABP认证;多聚腺苷酸结合蛋白，eIF4A：真核翻译起始因子4α，收缩压，eIF4E的;其余的TAP诱饵蛋白，含有融合的真核翻译起始因子4E，4EBPs;真核生物翻译起始因子4E结合的链亲和素的结合肽蛋白质，eIF4eNiF1l真核翻译起始因子4E核进口因子1，收缩压（SBP），其余的从TEV非稠合的TAP肽链霉结合肽。

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	<li>Williamson, M. P., Sutcliffe, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-protein+interactions.">Protein-protein interactions.</a> Biochem. Soc. Trans. 38 (4), 875-878 (2010).</li><li>Rigaut, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+generic+protein+purification+method+for+protein+complex+characterization+and+proteome+exploration.">A generic protein purification method for protein complex characterization and proteome exploration.</a> Nat. Biotechnol. 17 (10), 1030-1032 (1999).</li><li>Blackwell, C., Brown, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+application+of+tandem-affinity+purification+to+Candida+albicans.">The application of tandem-affinity purification to Candida albicans.</a> Methods Mol. Biol. 499, 133-148 (2009).</li><li>Cochrane, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+complex+formation+between+HIV+Rev+and+the+nucleosome+assembly+protein,+NAP1,+affects+Rev+function.">Stable complex formation between HIV Rev and the nucleosome assembly protein, NAP1, affects Rev function.</a> Virology. 388 (1), 103-111 (2009).</li><li>Fernandez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+tandem+affinity+purification+of+PSD-95+recovers+core+postsynaptic+complexes+and+schizophrenia+susceptibility+proteins.">Targeted tandem affinity purification of PSD-95 recovers core postsynaptic complexes and schizophrenia susceptibility proteins.</a> Mol. Syst. Biol. 5, 269-269 (2009).</li><li>Gloeckner, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tandem+affinity+purification+of+protein+complexes+from+mammalian+cells+by+the+Strep&#47;FLAG+(SF)-TAP+tag.">Tandem affinity purification of protein complexes from mammalian cells by the Strep&#47;FLAG (SF)-TAP tag.</a> Methods Mol. Biol. 564, 359-372 (2009).</li><li>Holowaty, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+profiling+with+Epstein-Barr+nuclear+antigen-1+reveals+an+interaction+with+the+herpesvirus-associated+ubiquitin-specific+protease+HAUSP/USP7.">Protein profiling with Epstein-Barr nuclear antigen-1 reveals an interaction with the herpesvirus-associated ubiquitin-specific protease HAUSP/USP7.</a> J. Biol. Chem. 278 (32), 29987-29994 (2003).</li><li>Burckstummer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+tandem+affinity+purification+procedure+for+interaction+proteomics+in+mammalian+cells.">An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells.</a> Nat. Methods. 3 (12), 1013-1019 (2006).</li><li>Goodfellow, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calicivirus+translation+initiation+requires+an+interaction+between+VPg+and+eIF+4+E.">Calicivirus translation initiation requires an interaction between VPg and eIF 4 E.</a> EMBO Rep. 6 (10), 968-972 (2005).</li><li>Goodfellow, I. G., Roberts, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eukaryotic+initiation+factor+4E.">Eukaryotic initiation factor 4E.</a> Int. J. Biochem. Cell Biol. 40 (12), 2675-2680 (2008).</li></ol>

没有利益冲突的声明。

说明这里的TAP标签技术演示为诱饵蛋白在真核细胞中分离的具有约束力的合作伙伴的高度特异性和严格的方法。这种方法可应用于细胞和病毒蛋白。据我们所知，这是第一次，这种技术已被应用到翻译起始因子eIF4E。知名eIF4E的结合蛋白eIF4G和使用这种技术的4EBPs鉴定，证实了这种方法的有效性。此外，鉴定eIF4F复杂的剩余部分，​​即eIF4A和PABP至证实，间接的互动关系和大专院校的复合物留在净化过程中的完整。 eIF4E的结合蛋白的典型多个亚型的识别也很明显。图2中更详细地描述了这些。 至于该方法的局限性，某些保健应采取的选择与问候诱饵蛋白和是否放置在N-或C-末端的TAG标签。这可能是最好进行功能检测，或本地化融合蛋白通过显微镜检查，以确保标签的衍生工具是功能完整的大规模纯化。积分膜或核蛋白质不一定被释放裂解步骤中所述的条件相对温和的洗涤剂。与所有免疫沉淀和类似拉下来分析，修改，可以在提高效率的裂解裂解液的性质和浓度的洗涤剂。离子浓度的溶解和洗涤缓冲也可以修改，增加或减少净化的严格。它也可以执行初步净化温和条件下标准（如上所述），但随后分裂的链亲和素珠（5.8节）为4-5等份，随后被冲下离子浓度增加ditions。这可能使用户能够确定的最佳条件，消除非特异性相互作用的蛋白质。整个过程中也可以使用互动的合作伙伴被称为瞬时转染和随后印迹确定其存在或不存在。在这种情况下，我们建议各8微克的表达质粒转染细胞100厘米2菜。这个修改​​过程是研究突变衍生物的TAP融合蛋白与已知的具有约束力的合作伙伴互动的能力时，特定用途。 分析样品1，通过银染SDS-PAGE凝胶（或印迹，如果抗体是可用的）8，是一个很好的方法排除故障，技术故障产生可接受的结果。使用此系统抽样的方法，可以分析的基于每个亲和力的降水和具体洗脱效率。样品应采取一至八个期间OPTIM化的协议，为每个新的诱饵。 TAP标签技术提供了一个强大和稳健的替代其他方法，如GST拉，酵母双杂交实验等双亲和纯化和具体洗脱步骤（TEV酶切和生物素洗脱）提供特异性和严格保持在整个净化过程中的较高水平。批判这种技术可以应用到任何蛋白质，因此确定为目标蛋白结合合作伙伴的一个极好的方法。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>潮霉素B </td><td>罗氏公司</td><td> 10843555001 </td></tr><tr><td>兔IgG的琼脂糖</td><td>西格玛</td><td> A2909 </td></tr><tr><td> AC-TEV蛋白酶</td><td> Invitrogen公司</td><td> 12575-015 </td></tr><tr><td>蛋白酶抑制剂</td><td> Calbiochem公司</td><td> 539134 </td></tr><tr><td> ULTRALINK链霉亲和素固定加珠</td><td>刺穿</td><td> 53116 </td></tr><tr><td> vivaspin 500离心选矿（5KDa） </td><td> vivaspin </td><td> V50112 </td></tr><tr><td> SilverQuest银染试剂盒</td><td> Invitrogen公司</td><td> LC6070 </td></tr><tr><td> NOVEX蓝色胶体考马斯染色试剂盒</td><td> Invitrogen公司</td><td> LC6025 </td></tr><tr><td> 1.7毫升预润滑管</td><td> COSTAR </td><td> 3207 </td></tr><tr><td>微毛细管移液器提示</td><td>厂商VWR </td><td> 37001-150 </td></tr><tr><td> NuPage 4-12％二，三梯度凝胶</td><td> Invitrogen公司</td><td> NP0322BOX </td></tr><tr><td> CDCL 2 </td><td>西格玛</td><td> 202908 </td></tr><tr><td> 5X SDS样品缓冲</td><td>费舍尔</td><td> PN39000 </td></tr></tbody></table>

identification of protein interacting partners using tandem affinity purification

一个经常限制的关键一步，在了解主机和病毒蛋白的功能是相互作用的细胞或病毒蛋白伙伴的标识。有许多方法，使识别的互动合作伙伴，包括酵母双杂交系统，以及下拉检测用重组蛋白免疫沉淀和内源性蛋白质质谱鉴定1。最近的研究都强调实用的双亲和力标签介导的净化，再加上两个相互作用的蛋白质鉴定的具体洗脱步骤。这种方法称为串联亲和纯化（TAP）的，最初用于酵母2,3，但最近已经适应在哺乳动物细胞中的使用4-8。 作为证据的概念，我们已经建立了串联亲和纯化（TAP）的方法，利用良好的特点真核翻译起始因子TOR eIF4E的9,10。细胞翻译因子eIF4E是一个蜂窝eIF4F复杂的重要组成部分，涉及帽依赖的翻译起始10。在目前的研究中使用的TAP标签是两个G蛋白单位组成，由烟草蚀纹病毒（TEV）蛋白酶切割序列1链亲和素结合肽的分离。在目前的研究中使用的TAP标签组成的两个G蛋白单位和1链亲和素结合肽的分离，由烟草蚀纹病毒（TEV）蛋白酶切割序列8。放弃代克隆细胞系的需要，我们开发了一种快速的系统依赖于从episomally保持质粒的TAP标签的诱饵在pMEP4（Invitrogen公司）为基础蛋白表达。使用氯化镉诱导金属硫蛋白启动子控制的标签从这个质粒的小鼠eIF4E的表达。 通过结合到R的表达和随后的亲和纯化的裂解abbit抗体琼脂糖，TEV蛋白酶裂解，结合链霉联系琼脂糖和生物素洗脱随后确定了许多蛋白质​​显然是具体的eIF4E的下拉（时相比，控制细胞株单独表达TAP标签）。蛋白质的身份获得了从一维的SDS-PAGE和随后的串联质谱带切除。确定的组件包括知名eIF4E的结合蛋白eIF4G和4EBP-1。此外，eIF4F复杂的其他组成部分，其中eIF4E的是一个组件被确定，即eIF4A和聚A结合蛋白。识别能力，不仅直接约束力的合作伙伴以及二级相互作用的蛋白质，进一步突出了在未知功能的蛋白质的特性，这种方法的效用。

Watch this Scientific Journal Video about Identification of Protein Interacting Partners Using Tandem Affinity Purification at JoVE.com

Identification of Protein Interacting Partners Using Tandem Affinity Purification

串联亲和纯化的蛋白结合合作伙伴的标识是稳健的做法。由于概念证明，这种方法的应用以及特色的翻译起始因子eIF4E共沉淀的宿主细胞因子参与翻译起始。这种方法是很容易适应任何细胞或病毒蛋白。

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-protein-interacting-partners-using-tandem-affinity

Research

JoVE Journal

Biology

37.3K 次观看.  Imperial College London. 串联亲和纯化的蛋白结合合作伙伴的标识是稳健的做法。由于概念证明，这种方法的应用以及特色的翻译起始因子eIF4E共沉淀的宿主细胞因子参与翻译起始。这种方法是很容易适应任何细胞或病毒蛋白。

视频: Identification of Protein Interacting Partners Using Tandem Affinity Purification

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 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 ...

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 and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

使用具体远藤和exoglycosidases糖基化蛋白质的鉴定和表征

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

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 ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

一个协议，用于基于蛋白质 - 蛋白质相互作用的鉴定<sup&gt; 15</sup&gt; N代谢标记，免疫沉淀法，的定量质谱法和亲和调制

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

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 ...