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不包含HE或其他限制性位点的正确的开放阅读框（ORFs）的存在下进行验证。考虑订购合成基因昆虫细胞密码子的使用和mRNA的二级结构进行了优化，以提高蛋白质的生产水平以及去除任何鄂西蜇HE网站感兴趣的基因。关于灵活或暴露的N-或C-末端蛋白选择从文献数据的基础上考虑将净化标签。考虑申请聚战略，着眼于生产多种蛋白质亚基在复杂，如果单个蛋白的相对量在复杂的由于化学计量问题，需要加以控制。4准备详细的"如何"文件（电子实验室本书推荐）预计该项目的实验步骤，完整的多基因结构（次）。建立电子档案的Cre-LoxP位融合质粒例如伯杰组<a href="http://www.embl.fr/multibac/%20multiexpression_technologies/cre-acembler" target="_blank">www.embl.fr/multibac/ multiexpression_technologies / CRE-acembler的</a>主页（ ）可以直接从网上下载使用CRE-ACEMBLER的软件。 </li><li>你感兴趣的基因插入到选定的捐助者和用限制性内切酶和连接酶，或者，通过使用独立结扎方法后发布的协议。受体5,6,14如果您有液体处理工作站的访问，如果你计划要产生大量的结构（例如组合方法）考虑使用机器人脚本伯杰组（ 图2）。14,15制定和实施，如果液体处理站工作，手动操作使用微孔板允许基因插入在HT喜欢时尚。 </li><li>需要表达亚基的化学计量控制所施加的约束可能会实现。在化学计量学上不均衡的蛋白质复合体的各个亚基的表达水平的情况下，可以考虑应用多聚策略相合由s隔开的单个大的ORF中的一些亚基的复杂和特定的蛋白酶（例如烟草蚀纹病毒的NIa蛋白酶）蛋白水解网站pecific 4,8考虑共同表达一个或多个多聚单表达盒，如果你有一个非常大的复杂许多亚基和广泛单个亚基分子量不等。考虑共同表示几个相同的蛋白质编码基因中的多聚或几个相同的表达盒的特征在于，如果该蛋白产品收率低。4,13 </li><li>验证所有的供体和受体的结构通过的限制映射（可选高通量）和测序克隆。继续的Cre-loxP重组融合给体 - 受体组合生成的多基因表达结构的选择。验证纯化接受器捐助者融合质粒的限制映射，使用创建的电子序列利用Cre-ACEMBLER的或类似的计划作为参考。 </li><li>纯化和验证的捐助者，受体和供体 - 受体融合存放在-20°C或-80°C。存档PLasmids序列（Microsoft Excel中，FILEMAKER，其他）仔细供以后使用。 </li></ol> 2。复合多基因杆状病毒代，扩增和存储<ol><li>整合多基因转移载体MultiBac的的杆状病毒基因组DH10细胞转化成窝藏病毒的基因组和TN7换位需要的功能。需要注意的是杆状病毒基因组Multibac由体内酶Cre反应前Tn7转集成在与感兴趣的特定基因（YFP标记，分子伴侣等）在其自己的LoxP位站点（工程远端Tn7转附着位点到基因组中），可以被预先加载。 13 TN7换位后复合杆状病毒包含的基因，细胞蓝/白筛选（成功TN7换位业绩亏损的β-半乳糖苷酶的α-互补，因此，保持白色菌落正确TN7换位选择性一个选择石榴石含有X-gal的平板）和基因组的制备采用碱裂解和乙醇/异丙醇沉淀。5,6 </li><li> 转染和最初的病毒生产 。广场6孔培养板放入无菌罩。从对数期的Sf21昆虫细胞培养，种子满分井和转染细胞的等分试样，加入纯化的杆状病毒基因组和混合在培养基中的转染试剂，如所述。6收获后48-60小时的初始病毒移除媒体（高品质，低滴度的病毒V 0，一般为3毫升，每孔）。补充新鲜培养基和测试6,7蛋白生产后2-3天（YFP标记如果存在，荧光）。 </li><li> 扩增病毒，低MOI方案 。使用V 0病毒感染25-50毫升对数生长期的细胞（细胞密度&lt;1×10 6细胞每毫升）小（100-250毫升）锥形瓶瓶激动的轨道平台呼风唤雨（ 图3）。计数细胞和细胞分裂每24个小时，直到停止倍增（增殖被捕）。按照低MOI（多重感染， 即每个细胞的病毒颗粒）的方案:细胞必须加倍（至少）一次（MOI &lt;1），否则重复实验，更小的体积V 0。通常情况下，V 0 3毫升用于感染的Sf21昆虫细胞中的25毫升的密度为0.5×10 6细胞/ ml。这是必要的，以防止有害的过度放大和自动删除病毒，可能导致的异源基因的利益损失。收获V 1病毒（25-50毫升）48-60小时后，通过制粒细胞和去除含有病毒的媒体。补充新鲜的培养基和试验，每12或24小时，除去（1×10 6个细胞），制粒和验证蛋白质的生产和标记蛋白（YFP）信号。6 YFP的蛋白质生产，病毒扩增（到V 2）进一步表达量较大的目的是通过感染2摇瓶400毫升细胞与V 1病毒尊重上述低MOI方案（细胞必须加倍至少一次感染后V 1）。严格测试蛋白质生产和标记蛋白信号在放大过程中避免积累不再含有您感兴趣的基因缺陷病毒。5-7使用细胞颗粒积聚在每个扩增步骤已经建立利益表达的蛋白复合物的净化协议。 </li><li> 生产病毒的BIIC存储 。我们强烈建议使用存储V 2病毒作为生产病毒BIIC（B aculovirus 我我 NSECT nfected的C ELL学生）的方法，以防止修改（ 如失去感兴趣的基因）的重组病毒，并保留高表达水平16颗粒感染细胞增殖逮捕后24小时观察-在这一阶段细胞含有完整的病毒颗粒之前，他们会被释放到媒体（萌芽）。取出介质，在液氮中冷冻等分的细胞沉淀，并存储7,16下去。 </li></ol> 3。蛋白质生产及下游加工<ol><li> 感染大（R）文化和监测YFP。使用V 1，V 2或冷冻BIIC等分感染的细胞培养物的生产运行（在2L烧瓶通常400毫升）。坚持低MOI方案（调整病毒感染感染培养双打至少一次的使用量）。烧瓶数乘以放大感染培养卷，如果需要。 YFP标记蛋白存在，退出定义的时间间隔为1×10 6细胞，颗粒和细胞溶解和监测YFP的信号演变，直至达到一个高原工业icating最大生产重组蛋白。 YFP的电平可以在一个标准的96孔板的读者能够记录荧光信号（ 例如，Tecan公司SPECTRAFluor）的测量。在这个阶段的收获细胞。细胞颗粒存储于-20℃（短期）或-80℃（长期）。 </li><li> 细胞裂解和分级分离 。您最喜爱的首选方法，蛋白质（冻融，超声，法国媒体，其他）的要求量身定做。裂解细胞5-7分馏细胞质和细胞核和测试您感兴趣的蛋白质的存在。开发提纯协议，成果的基础上，以简化蛋白质纯化。考虑申请浸泡程序，提取蛋白的核分数高氯化钾条件下，如果驻留在细胞核中的蛋白质。7,18 </li><li> 蛋白质纯化（微观尺度，大规模）。需要注意的是细胞培养的往往是小卷（10或25毫升）是苏fficient获得的细胞沉淀净化您感兴趣的蛋白质大量典型的高或非常高的异源蛋白的生产水平在杆状病毒/昆虫细胞系统（通常是10-100毫克每升的文化和更多的蛋白质）。 ，微净化（多孔板，微尖的方法，GE医疗ÄKTAmicro系统，其他的）相结合，这是可能的获得生化和活动的数据，常常也足够量的纳升规模的高通量结晶所需的蛋白质和复合物（HTX ）。暴露的蛋白复合物的亚单位，以便净化，连同小的离子交换和尺寸排阻色谱法，可以考虑使用金属亲和纯化（Qiagen公司的NiNTA CLONETECH宝TALON金属螯合剂树脂）和寡聚组氨酸（6-10个氨基酸残基）标签例如使用ÄKTAmicro的或类似的小体积净化机（ 图4卷</st荣&gt;）。其他亲和纯化步骤和离子交换（IEX）在除了与寡聚组氨酸标签或其他标记的亲和纯化步骤（GST，MBP，CBP，其他选择）即可，并应考虑，根据蛋白质的生化特性的兴趣和个人喜好。考虑标记一个以上的子单元与亲和标签，以提高净化效率。浓缩纯化的蛋白复合物，并建立存储协议，如冻结或不含甘油。制定质量控制标准，可以应用标准（活性​​试验，生物化学和生物物理测试），以评估纯化的蛋白质批次与批之间的变化。 </li></ol>

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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>

IB是在详细说明部分的技术，这里所描述的专利和专利申请的发明人。

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-CUBE，BioSTRUCT-X）的专线接入方案。 标准操作程序开展蛋白复合物的表达使用MultiBac系统的可用性，该技术也容易服从于非专业用户。操作机器人辅助加速的多蛋白复合生产partic特别当一个足够大的数量的复合物，例如，变体和突变体的检体还需要生成并行。然而，手动操作在低到中等吞吐量也大大受益从可用性的SOP。根据我们的经验，加以提炼过程，可能是成功的脚本程序到机器人需要先有显着的努力，直到获得足够强大的协议兼容使用机器人。这些协议的基础，我们的标准操作程序。5,6,14,15事实上，这些机器人的可靠的协议的实施导致一个非常可观的效率增益也手工操作在我们的实验室。 许多蛋白质​​和蛋白质复合物已经和正在生产使用MultiBac我们开发的系统，并且已经获得了近500名世界各地的实验室试剂。 MultiBac催化研究不仅在结构生物学，而且在许多超视距呃生命科学领域的研究或开发大型装配体中的蛋白质之间的相互作用。 MultiBac也被用来产生相当的药理利益目标蛋白的病毒样颗粒，这可能会成为有用的候选疫苗。MultiBac 4最近也被用于提供到哺乳动物细胞和细胞培养的基因，或者甚至整个生物体基因疗法。4，我们预计，那些在这方面的贡献所示的方法，比如将被证明是有用的多蛋白的组件和生物大分子的复杂的相互作用，形成了基础的健康和疾病的细胞过程的研究，涉及许多领域。

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）的基因组装实验的工作流程。鲁棒DNA组装协议都是照本宣科，多基因表达结构的并行组装成机器人例程。个人机器人的步骤单元镜头（ 图2c，I - IV）所示。进行组装的由PCR产生的DNA成分和质量控制的电子凝胶（ 图2d，左）;的组装多基因结构也同样通过PCR验证与专对引物（ 图2d，右）。14,15 重组杆状病毒的产生和放大遵循标准操作程序（示意图显示在图3a）。细胞单层培养的快照（ 图3b，I.＆II。）以下与MultiBac病毒感染。 可以小型化，通过使用多井板或基于微尖的色谱的亲和纯化，通过尺寸排阻色谱法（SEC）的配合物通过融入的工作流系统，如小规模的重组蛋白复合物的下游加工ÄKTAmicro（ 图4a）。 700 kDa的人类转录因子复杂的一位代表SEC档案显示。纯化的样品通过使用，小规模ÄKTAmicro通常是足够的表征生物化学和生物物理的手段，包括电子显微镜（ 图4b）。 <img alt="图1" src="/files/ftp_upload/50159/50159fig1.jpg" /> 图1。 MultiBac多蛋白复合生产平台技术。 （一）感兴趣的基因被整合到杆状病毒基因组Multibac通过Tn7转换位连同蓝/白筛选。病毒骨干甲LoxP位网站允许加入其它的功能，如荧光标记蛋白监测病毒的性能和生产异源蛋白质，（b）在杆状病毒采用一个细长的棒状，其特征在于，通过一个灵活的包络线，可以增加，以适应大（&gt; 130 KB）环状双链DNA基因组中。大基因组中的异源基因插入的耐受性bÝ伸长的包络，（三）在轨道摇床平台上的标准三角烧瓶中，可用于大规模昆虫细胞培养增长为heterologues蛋白质生产（d）本MultiBac系统可有效地生产重组蛋白，这往往是清晰可见的，已经在整个细胞提取物（SNP）。（五）有丝分裂关卡复杂的结构，阐明了从样品生产17 ​​的MultiBac系统<a href="https://www.jove.com/files/ftp_upload/50159/50159fig1large.jpg" target="_blank">点击此处查看大图</a>从晶体的X射线衍射。 <img alt="图2" src="/files/ftp_upload/50159/50159fig2.jpg" /> 图2。 （自动）串联重组工程（TR）。 （一）串联重组工程采用的合成质粒DNA分子，称为小数组组装多个供体和受体任选表达构建igene，在机器人辅助模式下使用的液体处理工位（右），（b）本TR的工作流程示出。 SLIC的代表序列和结扎独立克隆，CRE代表的Cre-LoxP位融合串联捐助者和接受者到感兴趣的基因已被插入SLIC。多基因表达构造，该重组工程的过程所产生的使用SLIC和中铁快运串联，然后融入MultiBac杆状病毒基因组，并用于小型和大型表达重组病毒感染昆虫细胞培养。（三）快照机器人辅助TR的过程中显示的模板DNA和引物（Ⅰ）在多孔板（II），（III）的DNA的PCR扩增和制备多基因的PCR反应，制备构建体，包括提供多孔板中采用碱裂解细菌培养中生长（四）。（四）我们可视化的PCR产物用于TRING电子凝胶系统（左）。已完成的多基因结构验证分析PCR反应一封凝胶（右）装上。 <a href="https://www.jove.com/files/ftp_upload/50159/50159fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" src="/files/ftp_upload/50159/50159fig3.jpg" /> 图3。 MultiBac病毒的产生，放大，存储。 （一）在EMBL格勒诺布尔MultiBac平台的标准操作程序（SOP）中所示的示意性视图。重组MultiBac病毒被确定蓝白斑筛选，并准备从细菌培养。最初的转染发生单层昆虫细胞（草地贪夜蛾Sf9昆虫，将hi5，其他的）接种在6孔板中。病毒被放大和靶蛋白的产生在锥形振动筛。病毒感染的昆虫细胞冷冻等分（BIIC）存储。作为一种分析工具，如果花期记录YFP（或其他荧光蛋白）已经被集成到MultiBac杆状病毒基因组的loxP位点上存在例如作为标记蛋白（二）所示MultiBac杆状病毒感染的昆虫细胞中的快照。细胞停止增殖，增加大小（一）。观察到细胞融合（II）。发芽关闭病毒感染的细胞进入媒体收集和使用的蛋白复合物的生产（III，IV）感染的细胞培养。 <a href="https://www.jove.com/files/ftp_upload/50159/50159fig3large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图4" src="/files/ftp_upload/50159/50159fig4.jpg" /> 图4。蛋白复合物的生产和下游加工。蛋白质复杂的样品已经可以方便地纯化，利用小型化的纯化方法，如多层或微尖小规模的初始细胞培养基于纯化，任选地连同小体积的色谱系统（左）。已经这些"分析"净化器运行（中）的情况下，产量足够的分析纯化的复杂的结构和功能，通过各种手段，包括电子显微镜（右） <a href="https://www.jove.com/files/ftp_upload/50159/50159fig4large.jpg" target="_blank">点击这里查看大图</a> 。

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 ...

the-multibac-protein-complex-production-platform-at-the-embl

Nanyang Technological University

Research

JoVE Journal

Biology

15.8K 次观看.  EMBL Grenoble Outstation and Unit of Virus Host Cell Interactions (UVHCI) UMR5322. 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。

视频: The MultiBac Protein Complex Production Platform at the EMBL

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of the stomach is defined by the mouth opening and the posterior end by the bottom of the pylorus. Along the dorsal side of the stomach lies the stomatogastric nervous system (STNS). This nervous system, which contains rhythmic networks that underlie feeding behavior, is an established model system for studying rhythm generating networks and neuromodulation 1,2. While it is possible to study this system in vivo 3, the STNS continues to produce its rhythmic activity when isolated in vitro. In order to study this system in vitro the stomach must be removed from the animal. This video article describes how the stomach can be dissected from the American lobster. In an accompanying video article4 we demonstrate how the STNS can be isolated from the stomach.

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

We describe the gross dissection of the stomach of the American 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

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 ...

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

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

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

The tissue microarray (TMA) technology provides the means for high-throughput analysis of multiple tissues and cells. The technique is used within the Human Protein Atlas project for global analysis of protein expression patterns in normal human tissues, cancer and cell lines. Here we present the assembly of 1 mm cores, retrieved from microscopically selected representative tissues, into a single recipient TMA block. The number and size of cores in a TMA block can be varied from approximately forty 2 mm cores to hundreds of 0.6 mm cores. The advantage of using TMA technology is that large amount of data can rapidly be obtained using a single immunostaining protocol to avoid experimental variability. Importantly, only limited amount of scarce tissue is needed, which allows for the analysis of large patient cohorts 1 2. Approximately 250 consecutive sections (4 &mu;m thick) can be cut from a TMA block and used for immunohistochemical staining to determine specific protein expression patterns for 250 different antibodies. In the Human Protein Atlas project, antibodies are generated towards all human proteins and used to acquire corresponding protein profiles in both normal human tissues from 144 individuals and cancer tissues from 216 different patients, representing the 20 most common forms of human cancer. Immunohistochemically stained TMA sections on glass slides are scanned to create high-resolution images from which pathologists can interpret and annotate the outcome of immunohistochemistry. Images together with corresponding pathology-based annotation data are made publically available for the research community through the Human Protein Atlas portal (<a href="http://www.proteinatlas.org" target="_blank">www.proteinatlas.org</a>) (Figure 1) 3 4. The Human Protein Atlas provides a map showing the distribution and relative abundance of proteins in the human body. The current version contains over 11 million images with protein expression data for 12.238 unique proteins, corresponding to more than 61% of all proteins encoded by the human genome.

Tissue microarrays allows for an efficient method to gain concurrent information from a multitude of tissues. Representative parts of tissues are assembled into a single paraffin block. Sections from the block are used for immunohistochemistry and analysis of protein expression patterns. Digital scanning generates corresponding images for distribution of data.

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

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

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 heterologous production of complex natural products is an approach designed to address current limitations and future possibilities. It is particularly useful for those compounds which possess therapeutic value but cannot be sufficiently produced or would benefit from an improved form of production. The experimental procedures involved can be subdivided into three components: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each experimental component is under continual optimization to meet the challenges and anticipate the opportunities associated with this emerging approach.
Heterologous biosynthesis begins with the identification of a genetic sequence responsible for a valuable natural product. Transferring this sequence to a heterologous host is complicated by the biosynthetic pathway complexity responsible for product formation. The antibiotic erythromycin A is a good example. Twenty genes (totaling &gt;50 kb) are required for eventual biosynthesis. In addition, three of these genes encode megasynthases, multi-domain enzymes each ~300 kDa in size. This genetic material must be designed and transferred to E. coli for reconstituted biosynthesis. The use of PCR isolation, operon construction, multi-cystronic plasmids, and electro-transformation will be described in transferring the erythromycin A genetic cluster to E. coli.
Once transferred, the E. coli cell must support eventual biosynthesis. This process is also challenging given the substantial differences between E. coli and most original hosts responsible for complex natural product formation. The cell must provide necessary substrates to support biosynthesis and coordinately express the transferred genetic cluster to produce active enzymes. In the case of erythromycin A, the E. coli cell had to be engineered to provide the two precursors (propionyl-CoA and (2S)-methylmalonyl-CoA) required for biosynthesis. In addition, gene sequence modifications, plasmid copy number, chaperonin co-expression, post-translational enzymatic modification, and process temperature were also required to allow final erythromycin A formation.
Finally, successful production must be assessed. For the erythromycin A case, we will present two methods. The first is liquid chromatography-mass spectrometry (LC-MS) to confirm and quantify production. The bioactivity of erythromycin A will also be confirmed through use of a bioassay in which the antibiotic activity is tested against Bacillus subtilis. The assessment assays establish erythromycin A biosynthesis from E. coli and set the stage for future engineering efforts to improve or diversify production and for the production of new complex natural compounds using this approach.

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

The heterologous biosynthesis of erythromycin A through E. coli includes the following experimental steps: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each step will be explained in the context of the motivation, potential, and challenges in producing therapeutic natural products using E. coli as a surrogate host.

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

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

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

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

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

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

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

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

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

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

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 ...

在EMBL的MultiBac蛋白质复杂的生产平台

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肚子里的龙虾总夹层， Homarus Americanus

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

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

一个从人体细胞分泌蛋白生产的方便，一般的表达平台

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

人鼻粘膜上皮细胞在气液界面培养

定量免疫检测，以衡量在中心体的变异蛋白水平

膜蛋白的绿色荧光蛋白为基础的表达筛选

CUBIC协议可视化在全山皮肤配方单细胞分辨率蛋白表达

在EMBL的MultiBac蛋白质复杂的生产平台

摘要

探索更多视频

此视频中的章节

肚子里的龙虾总夹层， Homarus Americanus

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

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

一个从人体细胞分泌蛋白生产的方便，一般的表达平台

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

人鼻粘膜上皮细胞在气液界面培养

定量免疫检测，以衡量在中心体的变异蛋白水平

膜蛋白的绿色荧光蛋白为基础的表达筛选

CUBIC协议可视化在全山皮肤配方单细胞分辨率蛋白表达

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