Name: the multibac protein complex production platform at the embl
Uploaded: 2013-07-11T12:00:00
Description: Watch this Scientific Journal Video about The MultiBac Protein Complex Production Platform at the EMBL at JoVE.com

我们感谢丹尼尔·菲茨杰拉德，西门Trowitzsch，克里斯托夫Bieniossek，高木雄一郎，Christiane Schaffitzel的，伊冯娜亨泽尔，蒂莫西·里士满和所有过去和现在的成员伯杰实验室寻求帮助和建议。 MultiBac平台，其发展已经和慷慨支持资助机构包括瑞士国家科学基金会（瑞士国家基金会），法新社巴黎国家（ANR）和巴黎国家科学研究中心（CNRS）和欧洲委员会（EC）在框架程序（FP）6和7。支持跨国的访问是由欧盟第七框架计划项目P-CUBE（ <a href="http://www.p-cube.eu" target="_blank">www.p-cube.eu</a> ）和BioStruct-X（ <a href="http://www.biostruct-x.eu" target="_blank">www.biostruct x.eu</a> ）。法国科技部特别承认用于支持MultiBac EMBL的平台通过的INVESTISSEMENT d&#39;艾文莉项目FRISBI。

1。串联重组工程（TR）用于创建多基因表达结构<ol><li> 规划的共表达策略 。你感兴趣的基因插入到供体和受体的设计方法。您的复杂的潜在生理子模块应集中在特定的接受者和捐助者。用乘法模块组成的归巢核酸内切酶（HE） - BstXI位对个人的供体和受体质粒表达盒结合7,8建立在硅片和所有相关的构造彻底验证策略，然后再进行试点工作。例如，感兴趣的基因应检查?...

<ol>
	<li>Nie, Y., Viola, C., Bieniossek, C., Trowitzsch, S., Vijay-Achandran, L. S., Chaillet, M., Garzoni, F., Berger, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+a+Grip+on+Complexes.">Getting a Grip on Complexes.</a> Curr. Genomics. 10 (8), 558-572 (2009).</li><li>Robinson, C. V., Sali, A., Baumeister, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+sociology+of+the+cell.">The molecular sociology of the cell.</a> Nature. 450 (7172), 973-982 (2007).</li><li>Kost, T. A., Condreay, J. P., Jarvis, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus+as+versatile+vectors+for+protein+expression+in+insect+and+mammalian+cells.">Baculovirus as versatile vectors for protein expression in insect and mammalian cells.</a> Nat. Biotechnol. 23 (5), 567-575 (2005).</li><li>Bieniossek, C., Imasaki, T., Takagi, Y., Berger, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MultiBac:+expanding+the+research+toolbox+for+multiprotein+complexes.">MultiBac: expanding the research toolbox for multiprotein complexes.</a> Trends Biochem. Sci. 37 (2), 49-57 (2012).</li><li>Fitzgerald, D. J., Berger, P., Schaffitzel, C., Yamada, K., Richmond, T. J., Berger, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+complex+expression+by+using+multigene+baculoviral+vectors.">Protein complex expression by using multigene baculoviral vectors.</a> Nat. Methods. 3 (12), 1021-1032 (2006).</li><li>Bieniossek, C., Richmond, T. J., Berger, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MultiBac:+multigene+baculovirus-based+eukaryotic+protein+complex+production.">MultiBac: multigene baculovirus-based eukaryotic protein complex production.</a> Curr. Protoc. Protein Sci. Chapter 5, Unit 5.20 (2008).</li><li>Trowitzsch, S., Bieniossek, C., Nie, Y., Garzoni, F., Berger, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+baculovirus+expression+tools+for+recombinant+protein+complex+production.">New baculovirus expression tools for recombinant protein complex production.</a> J. Struct. Biol. 172 (1), 45-54 (2010).</li><li>Vijayachandran, L. S., Viola, C., Garzoni, F., Trowitzsch, S., Bieniossek, C., Chaillet, M., Schaffitzel, C., Busso, D., Romier, C., Poterszman, A., Richmond, T. J., Berger, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robots,+pipelines,+polyproteins:+enabling+multiprotein+expression+in+prokaryotic+and+eukaryotic+cells.">Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells.</a> J. Struct. Biol. 175 (2), 198-208 (2011).</li><li>Thomas, M. C., Chiang, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+general+transcription+machinery+and+general+cofactors.">The general transcription machinery and general cofactors.</a> Crit. Rev. Biochem. Mol. Biol. 41 (3), 105-178 (2006).</li><li>Klinge, S., Voigts-Hoffmann, F., Leibundgut, M., Ban, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+structures+of+the+eukaryotic+ribosome.">Atomic structures of the eukaryotic ribosome.</a> Trends Biochem. Sci. 37 (5), 189-198 (2012).</li><li>Melnikov, S., Ben-Shem, A., Garreau de Loubresse, N., Jenner, L., Yusupova, G., Yusupov, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+core,+two+shells:+bacterial+and+eukaryotic+ribosomes.">One core, two shells: bacterial and eukaryotic ribosomes.</a> Nat. Struct. Mol. Biol. 19 (6), 560-567 (2012).</li><li>Cramer, P., Bushnell, D. A., Fu, J., Gnatt, A. L., Maier-Davis, B., Thompson, N. E., Burgess, R. R., Edwards, A. M., David, P. R., Kornberg, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+RNA+polymerase+II+and+implications+for+the+transcription+mechanism.">Architecture of RNA polymerase II and implications for the transcription mechanism.</a> Science. 288 (5466), 640-649 (2000).</li><li>Berger, I., Fitzgerald, D. J., Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus+expression+system+for+heterologous+multiprotein+complexes.">Baculovirus expression system for heterologous multiprotein complexes.</a> Nat. Biotechnol. 22 (12), 1583-1587 (2004).</li><li>Bieniossek, C., Nie, Y., Frey, D., Olieric, N., Schaffitzel, C., Collinson, I., Romier, C., Berger, P., Richmond, T. J., Steinmetz, M. O., Berger, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+unrestricted+multigene+recombineering+for+multiprotein+complex+production.">Automated unrestricted multigene recombineering for multiprotein complex production.</a> Nat. Methods. 6 (6), 447-450 (2009).</li><li>Nie, Y., Bieniossek, C., Frey, D., Olieric, N., Schaffitzel, C., Steinmetz, M. O., Berger, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ACEMBLing+multigene+expression+constructs+by+recombineering.">ACEMBLing multigene expression constructs by recombineering.</a> Nat. Protocols. , (2009).</li><li>Wasilko, D. J., Lee, S. E., Stutzman-Engwall, K. J., Reitz, B. A., Emmons, T. L., Mathis, K. J., Bienkowski, M. J., Tomasselli, A. G., Fischer, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H.D.+titerless+infected-cells+preservation+and+scale-up+(TIPS)+method+for+large-scale+production+of+NO-sensitive+human+soluble+guanylate+cyclase+(sGC)+from+insect+cells+infected+with+recombinant+baculovirus.">H.D. titerless infected-cells preservation and scale-up (TIPS) method for large-scale production of NO-sensitive human soluble guanylate cyclase (sGC) from insect cells infected with recombinant baculovirus.</a> Protein Expr. Purif. 65 (2), 122-132 (2009).</li><li>Chao, W. C., Kulkarni, K., Zhang, Z., Kong, E. H., Barford, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+mitotic+checkpoint+complex.">Structure of the mitotic checkpoint complex.</a> Nature. 484 (7393), 208-213 (2012).</li><li>Yamada, K., Frouws, T. D., Angst, B., Fitzgerald, D. J., DeLuca, C., Schimmele, K., Sargent, D. F., Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+mechanism+of+the+chromatin+remodelling+factor+ISW1a.">Structure and mechanism of the chromatin remodelling factor ISW1a.</a> Nature. 472 (7344), 448-453 (2011).</li></ol>

视频管理单元射击在图2和图3中示出的整个过程，从机器人辅助发电的多基因表达的cDNA，构建感染的昆虫细胞培养生产蛋白质的方式。已经开发了新的试剂（质粒和病毒）和可靠的协议，使管道依靠的SOP。整个管道已经实现在EMBL格勒诺布尔作为一个平台技术。 MultiBac平台已访问许多来自学术界和工业界的科学家所从事的多蛋白的研究。培训访问支持由欧盟委员会资助（P-...

控制生物活性蛋白质和其他生物分子采取一致行动，促进细胞的功能组件。著名的例子包括机械，转录成信使RNA的DNA中包含的遗传信息。在人类中，超过100种蛋白质在界定和规范的过程中走到一起转录基因，是形成大的复合物，10多亚基RNA聚合酶II和一般转录因子如TFIID，TFIIH和其他9其他的例子包括核糖体，由许多蛋白质​​和RNA分子，催化蛋白质的合成，或者是负责穿梭生物分子通过在真核细胞的核膜核孔复合。一份详细的建筑和生化基本上所有多元的机器在细胞解剖，以了解它们的功能是至关重要的。的原核生物和eukar的结构鉴定例如，核糖体yotic，构成产生前所未有的洞察这些大分子机器如何开展其指定的功能，在细胞的标志性事件。10,11 核糖体，可以得到足够的质量和数量进行详细研究中，通过纯化从培养细胞中的内源性物质，由于这样的事实，细胞质量的30％，由核糖体。 RNA聚合酶II已经是不太丰富的量级，和几千升酵母培养物处理，以取得详细的原子鉴于这种必需的复杂的中央转录12然而目前在绝大多数其他必要的配合低得多的原生细胞，因而不能充分纯化从本地源材料。为了呈现这种复合访问详细的结构和功能分析，需要利用异源生产重组德chniques。 重组蛋白的生产产生了重大影响，对生命科学的研究。许多蛋白质​​是重组产生的，并且它们的结构和功能，在高分辨率解剖。结构基因组学计划已利用阐明整个生物高通量（HT）模式来解决基因产物的剧目很多生物的基因组。数以千计的蛋白质结构已被确定。到今天为止，最力强的重组蛋白生产系统已经E.多年来在此主机中的异源生产的大肠杆菌 ，以及许多表达系统已被开发和高雅。质粒窝藏了大量的功能，使蛋白质在大肠杆菌中生产大肠杆菌填满整个商业供应商目录。 然而，E。大肠杆菌具有一定的局限性，这使得它不适合产生许多真核生物蛋白在p关节与许多亚基的蛋白质复合物。因此，在真核宿主中的蛋白质生产方法的选择在最近几年已经变得越来越。一种特别适合的系统产生的真核生物蛋白是杆状病毒表达载体系统（BEVS），它依赖于携带异源基因的重组杆状病毒感染昆虫细胞培养在实验室中培养。 MultiBac系统是一个特别适合用于生产许多亚单位（ 图1）的真核细胞的蛋白质复合物，最近开发的BEVS。 MultiBac MultiBac在2004年首次推出13自推出以来，已不断完善和流内衬简化处理，提高目标蛋白的质量，一般非专业用户访问系统通过设计有效的标准操作程序（SOP）。在许多世界各地的实验室，交流已实施4 MultiBacademia和行业。在格勒诺布尔EMBL的，跨国的访问计划落实到位，由欧盟委员会提供专家培训在MultiBac平台，科学家们希望推进他们的研究，使用这种生产系统。迄今无法访问的多蛋白复合物的结构和功能鉴定，通过用MultiBac样品。4在下文中，总结于MultiBac生产的基本步骤的协议，因为它们是在操作在EMBL格勒诺布尔MultiBac设施。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Bluo-Gal</td>
 <td>Invitrogen</td>
 <td>15519-028 (1 g)</td>
 <td></td>
 </tr>
 <tr>
 <td>Tetracycline</td>
 <td>Euromedex</td>
 <td>UT2965-B (25 g)</td>
 <td>1,000X at 10 mg/ml</td>
 </tr>
 <tr>
 <td>Kanamycine</td>
 <td>Euromedex</td>
 <td>EU0420 (25 g)</td>
 <td>1,000X at 50 mg/ml</td>
 </tr>
 <tr>
 <td>Gentamycine</td>
 <td>SIGMA</td>
 <td>G3632 (5 g)</td>
 <td>1,000X at 10 mg/ml</td>
 </tr>
 <tr>
 <td>IPTG</td>
 <td>Euromedex</td>
 <td>EU0008-B (5 g)</td>
 <td>1,000X at 1M</td>
 </tr>
 <tr>
 <td>Cre-recombinase</td>
 <td>New England BioLabs</td>
 <td>M0298</td>
 <td></td>
 </tr>
 <tr>
 <td>X-Treme GENE HP transfection reagent</td>
 <td>Roche</td>
 <td>06 366 236 001</td>
 <td></td>
 </tr>
 <tr>
 <td>Hyclone SFM4 Insect</td>
 <td>Thermo Scientific</td>
 <td>SH 30913.02</td>
 <td></td>
 </tr>
 <tr>
 <td>6-well plate Falcon</td>
 <td>Dominique Dutscher</td>
 <td>353046</td>
 <td></td>
 </tr>
 <tr>
 <td>2 ml pipette Falcon</td>
 <td>Dominique Dutscher</td>
 <td>357507</td>
 <td></td>
 </tr>
 <tr>
 <td>5 ml pipette Falcon</td>
 <td>Dominique Dutscher</td>
 <td>357543</td>
 <td></td>
 </tr>
 <tr>
 <td>10 ml pipette Falcon</td>
 <td>Dominique Dutscher</td>
 <td>357551</td>
 <td></td>
 </tr>
 <tr>
 <td>25 ml pipette Falcon</td>
 <td>Dominique Dutscher</td>
 <td>357535</td>
 <td></td>
 </tr>
 <tr>
 <td>50 ml pipette Falcon</td>
 <td>Dominique Dutscher</td>
 <td>357550</td>
 <td></td>
 </tr>
 <tr>
 <td>50 ml tube Falcon</td>
 <td>Dominique Dutscher</td>
 <td>352070</td>
 <td></td>
 </tr>
 <tr>
 <td>15 ml tube Falcon</td>
 <td>Dominique Dutscher</td>
 <td>352096</td>
 <td></td>
 </tr>
 <tr>
 <td>1.8 ml cryotube Nunc</td>
 <td>Dominique Dutscher</td>
 <td>55005</td>
 <td></td>
 </tr>
 <tr>
 <td>100 ml shaker flasks Pyrex</td>
 <td>Dominique Dutscher</td>
 <td>211917</td>
 <td></td>
 </tr>
 <tr>
 <td>250 ml shaker flasks Pyrex</td>
 <td>Dominique Dutscher</td>
 <td>211918</td>
 <td></td>
 </tr>
 <tr>
 <td>500 ml shaker flasks Pyrex</td>
 <td>Dominique Dutscher</td>
 <td>211919</td>
 <td></td>
 </tr>
 <tr>
 <td>2 L shaker flasks Pyrex</td>
 <td>Dominique Dutscher</td>
 <td>211921</td>
 <td></td>
 </tr>
 <tr>
 <td>Certomat Orbital Shaker + plateau</td>
 <td>Sartorius</td>
 <td>4445110, 4445233</td>
 <td></td>
 </tr>
 <tr>
 <td>Liquid nitrogen tank dewar 35 L</td>
 <td>Fisher Scientific</td>
 <td>M76801</td>
 <td></td>
 </tr>
 <tr>
 <td>Biological Safety Cabinet Faster</td>
 <td>Sodipro</td>
 <td>FASV20000606</td>
 <td></td>
 </tr>
 <tr>
 <td>Optical Microscope</td>
 <td>Zeiss</td>
 <td>451207</td>
 <td></td>
 </tr>
 <tr>
 <td>Sf21 Insect cells</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Hi5 Insect cells</td>
 <td>Invitrogen</td>
 <td>B855-02</td>
 <td></td>
 </tr>
 <tr>
 <td>Tecan freedom EVO running Evoware plus</td>
 <td>TECAN</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>10 &mu;l conductive tips (black),</td>
 <td>TECAN</td>
 <td>10 612 516</td>
 <td></td>
 </tr>
 <tr>
 <td>200 &mu;l conductive tips (black)</td>
 <td>TECAN</td>
 <td>10 612 510</td>
 <td></td>
 </tr>
 <tr>
 <td>disposable trough for reagents, 100 ml</td>
 <td>TECAN</td>
 <td>10 613 049</td>
 <td></td>
 </tr>
 <tr>
 <td>twin.tec PCR plate 96, skirted</td>
 <td>Eppendorf</td>
 <td>0030 128.648</td>
 <td></td>
 </tr>
 <tr>
 <td>96 well V bottom, non sterile</td>
 <td>BD falcon</td>
 <td>353263</td>
 <td></td>
 </tr>
 <tr>
 <td>96 deepwell plate color natural, PP)</td>
 <td>Fisher</td>
 <td>M3752M</td>
 <td></td>
 </tr>
 <tr>
 <td>PS microplate, 96 well flat bottom</td>
 <td>Greiner</td>
 <td>655101</td>
 <td></td>
 </tr>
 <tr>
 <td>96 deepwell plate</td>
 <td>Thermo scientific</td>
 <td>AB-0932</td>
 <td></td>
 </tr>
 <tr>
 <td>24 well blocks RB</td>
 <td>Qiagen</td>
 <td>19583</td>
 <td></td>
 </tr>
 <tr>
 <td>DpnI restriction enzyme</td>
 <td>NEB</td>
 <td>R0176L</td>
 <td>20 U/uL</td>
 </tr>
 <tr>
 <td>NEBuffer 4 10X</td>
 <td>NEB</td>
 <td>B7004S</td>
 <td></td>
 </tr>
 <tr>
 <td>2X phusion mastermix HF</td>
 <td>Finnzyme</td>
 <td>ref F-531L</td>
 <td></td>
 </tr>
 <tr>
 <td>2X phusion mastermix GC</td>
 <td>Finnzyme</td>
 <td>ref F-532L</td>
 <td></td>
 </tr>
 <tr>
 <td>DGLB 1.5X</td>
 <td>homemade</td>
 <td></td>
 <td>7.5% glycerol, 0.031% Bromophenol blue, 0.031% Xylen cyanol FF</td>
 </tr>
 <tr>
 <td>High DNA Mass Ladder for e-gel</td>
 <td>Life Technologies</td>
 <td>10496-016</td>
 <td></td>
 </tr>
 <tr>
 <td>Low DNA Mass Ladder for e-gel</td>
 <td>Life Technologies</td>
 <td>10068-013</td>
 <td></td>
 </tr>
 <tr>
 <td>E-gel 48 1% agarose GP</td>
 <td>Life Technologies</td>
 <td>G8008-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Nucleo Spin- robot-96 plasmid kit</td>
 <td>Macherey Nagel</td>
 <td>740 708.24</td>
 <td></td>
 </tr>
 <tr>
 <td>PCR clean-up kit, Nucleospin Robot-96 Extract</td>
 <td>Macherey Nagel</td>
 <td>740 707.2</td>
 <td></td>
 </tr>
 <tr>
 <td>Gotaq green master mix</td>
 <td>Promega</td>
 <td>M7113</td>
 <td></td>
 </tr>
 <tr>
 <td>T4 DNA polymerase, LIC-qualified</td>
 <td>Novagen</td>
 <td>70099-3</td>
 <td></td>
 </tr>
 <tr>
 <td>DTT 100 mM</td>
 <td>homemade</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Urea 2 M</td>
 <td>homemade</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>EDTA 500 mM pH 8.0</td>
 <td>Homemade</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>LB broth (Miller) 500 g</td>
 <td>Athena ES</td>
 <td>103</td>
 <td></td>
 </tr>
 </tbody>
</table>

the multibac protein complex production platform at the embl

蛋白质组学的研究揭示了令人印象深刻的复杂的真核蛋白质组前所未有的细节。它现在是一个普遍接受的概念，细胞中的蛋白质主要存在不是孤立的实体，但发挥其生物活性协会与许多其他蛋白，在人类十个或更多的大多数，如果不是所有的重要职能，在细胞中形成组装线1。 ，2这些多蛋白组件的功能和体系结构的知识，需要他们提供卓越的质量和足够的数量进行详细的分析。细胞的多蛋白复合物的缺乏，特别是在真核生物中，禁止其提取从本地源，必须重组生产。杆状病毒表达载体系统（BEVS）已被证明是特别有用的真核蛋白质的制造，其中的活性往往依赖于翻译后加工等常用的表达系统通常可以不支持的。3的 BEVS使用到其中的感兴趣的基因插入的重组杆状病毒感染昆虫细胞培养物，这反过来又产生蛋白质给出。 MultiBac是一个BEVS已特别专为生产的真核细胞的蛋白质复合物包含许多亚基4高效生产的蛋白质和它们的配合物的一个重要的先决条件是，理想的情况下可实现为在表达式中实验所涉及的所有步骤的可靠的协议标准操作程序（SOP），随后也由非专业用户比较容易。 MultiBac在欧洲分子生物学实验室（EMBL）的平台使用的SOP在多蛋白复合表达实验中所涉及的所有步骤，从基因插入杆状病毒基因组的小规模分析的蛋白质的异源蛋白质的生产性能优化的一个精心设计的标本制作。5-8平台安装在EMBL格勒诺布尔在一个开放获取模式，并支持许多来自学术界和产业加速蛋白复合物的研究项目的科学家。

斯特朗合作MultiBac系统所实现的异源蛋白质的表达示于图1d（探针感染细胞悬浮培养后48小时）。在整个细胞提取物（SNP）和澄清的裂解液（SN）的过表达蛋白条带清晰可见。所产生的蛋白原料的质量和数量往往是足以使图1e中所示的结构测定的蛋白质复合物，例如有丝分裂关卡复杂MCC 17 图2显示了由机器人辅助串联重组工程（TR）?...

Watch this Scientific Journal Video about The MultiBac Protein Complex Production Platform at the EMBL at JoVE.com

The MultiBac Protein Complex Production Platform at the EMBL

蛋白质复合物催化关键的细胞功能。详细的功能及结构表征多种人体所需的复合需要重组生产。 MultiBac杆状病毒/昆虫细胞表达真核蛋白质及其复合系统特别定制。 MultiBac实施作为一个开放的接入平台，开发的标准作业程序，以最大限度地发挥其效用。

在EMBL的MultiBac蛋白质复杂的生产平台

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Research

JoVE Journal

Biology

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

肚子里的龙虾总夹层， Homarus Americanus</em

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of ...

科研

生物学

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

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 ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas

组织芯片，​​免疫组织化学染色和数字化生产在人类蛋白质图集

The  tissue microarray (TMA) technology provides the means for high-throughput  analysis of multiple tissues and cells. The technique is used within the ...

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

一个从人体细胞分泌蛋白生产的方便，一般的表达平台

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

的逻辑，实验步骤和外源天然产物生物合成的潜力，拥有复杂的抗生素红霉素通过<em&gt; E.大肠杆菌</em

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities.  It is ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

人鼻粘膜上皮细胞在气液界面培养

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

定量免疫检测，以衡量在中心体的变异蛋白水平

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

CUBIC协议可视化在全山皮肤配方单细胞分辨率蛋白表达

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...