这项工作是由安大略省的艾滋病毒治疗网络研究工作津贴（的ROG-G645）和加拿大健康研究的新研究者奖（的MSH-113554）研究院JEL和多伦多大学的奖学金支持HA，FCA和纪委。笔者想感谢在斯克里普斯研究所（拉霍亚加州）马妮福斯科，Dafna先生Abelson和埃里卡Ollmann萨费尔博士提供细胞，ebolavirus; GP表达载体和一般建议。

1。准备工作 - 构造和细胞培养在开始的协议之前，应该感兴趣的基因密码子优化，在哺乳动物细胞中表达，并克隆到一个合适的表达载体，利用标准的分子生物学技术。为了确保最高的成功表达的机会，应产生多个感兴趣的基因变种。许多哺乳动物表达载体可商业化，并有各种净化标签（多聚组氨酸，血凝素，链，晕标签，谷胱甘肽S-转移，等等）。我们更倾向于使用pDISPLAY载体，强大的人类巨细胞病毒启动子，免疫球蛋白κ分泌信号，血凝素纯化标签编码，并拥有C-末端的跨膜锚定目标蛋白通过细胞膜上显示的分泌途径。我们通常插入一个终止密码子在前面的向量编码的跨膜安乔r以使蛋白质分泌到条件培养基。 人胚肾293T细胞（HEK）被广泛使用，容易培养和转染。的HEK 293T通常用于哺乳动物蛋白的表达，但被认为是生物危害性，并应在生物安全2级处理。请穿着适当的个人防护服装;工作应在批准的生物安全柜使用无菌技术进行。根据体制和政府的方针，所有废物和表面应进行消毒处理。据建议，细胞被支原体污染，在使用前进行测试。细胞可被视为与环丙沙星（10微克/毫升）为10天，以消除任何支原体菌源。污染。 传播HEK 293T细胞的总协议单独列示（1盒）。 测试和大规模蛋白表达的其他注意事项reviewe在11-15。 2。小规模试验的表达一旦结构已经设计和产生的，小规模的试验转染HEK 293T细胞进行总结的过程示意图如下（ 图1）。 <ol><li>使用的T75 2厘米或T225 2厘米的细胞培养瓶（取决于进行测试表达式）增长HEK 293T细胞分裂的细胞时，细胞是100％的融合（1盒 ）每2-3天。 </li><li>种子2.5×10 5的HEK 293T细胞在6孔板，每孔与1X笔/链球菌和10％（V / V）2毫升培养液添加胎牛血清，旋流板轻轻地，以确保每口井，即使在细胞扩散， 5％的CO 2湿盒中孵育37℃过夜。 </li><li> HEK 293T细胞达到40％汇合时，丢弃的媒体与的1X笔/链球菌和10％（V / V）添加淡水2毫升培养液，胎牛血清的井。演出转染实验。 </li><li> 90μL无血清培养液分装到无菌的1.5ml离心管。吸取3μL的GeneJuice到无血清DMEM，轻轻地混合管（手指涡）。在室温下孵育5分钟。 </li><li>新增1微克小量纯化的质粒DNA（DNA的股票= 100毫微克/微升）为DMEM-GeneJuice混合物，手指漩涡，孵育15分钟，在室温下。 </li><li>吸取到HEK 293T细胞转染混合物滴加，漩涡6孔板轻轻地甚至允许转染混合物的分布。在37°C，5％的CO 2湿盒中孵育6孔板。 </li><li>加入1毫升1X笔/链球菌新鲜培养液和10％（V / V）FBS每口井的24小时内转染后48小时的孵育（共72小时）。 </li><li>收获1毫升上清从每口井在3天转染和离心10分钟，在室温下16000克样本。开展WES燕鸥在专栏2详细blot分析。样品可以储存在4°C存储长度为4°C的蛋白依赖。 </li></ol> 3。大规模试验的表达和纯化一旦结构已被确定毫克数量表达重组蛋白实现裴粘附使用10层的细胞工厂（ 图2;6360厘米2表面积）HEK 293T细胞转染的。对于更多的探索性研究，小细胞工厂或T-瓶（ 见表1）可以使用。 <ol><li>净化转，使用MaxiPrep质粒纯化试剂盒1毫克的DNA。 XL-1蓝细胞500毫升过夜培养，应该至少产生1毫克的纯DNA。检查DNA的纯度，通过测量A 260 / A 280比值应为1.8以上。 </li><li> HEK 293T细胞扩展到2.0×10 8个细胞。每个T225厘米2瓶增长到100％汇合合作ntains和〜2.25×10 7细胞的平均。 </li><li>新增1.2升5％（V / V）FBS DMEM培养液到10层的电池厂。加入2.0×10 8 HEK 293T细胞的细胞工厂和细胞均匀地分布到所有的船只层。这是非常困难的，可视化的细胞在细胞工厂的汇合。作为替代方案，设立适当数量的细胞1的T75厘米2瓶，使用相同的细胞数量，面积比为10层的船只进行。监测这一增长率瓶。参见处理电池厂指令相关视频。与5％CO 2孵育过夜，使细胞附着和生长，在37°C。 </li><li>附着HEK 293T细胞70％汇合时，执行大规模的转染。在生物安全柜使用无菌的T75厘米2瓶，准备的PEI-DNA转染混合物（3:1 W / W裴DNA比例）。 0.84毫克的DNA，用84毫升无菌1×PBS混合，再加入2.5毫升的PEI（2。5毫克总裴）。在室温下孵育15分钟。解决方案应该变得浑浊。 </li><li>进入细胞工厂的PEI-DNA转染混合物慢慢倒入和彻底分发到所有的船只层。可选:为表达增加产量，增加丙戊酸（终浓度为4毫米）。在37°C孵育5％的CO 2四天。 </li><li>收获上清转染4天。离心30分钟空调媒体4日在6000×Ğ°C。进一步筛选上清使用0.22微米Stericup的真空过滤装置。可重复使用10层的细胞工厂，清洁指示框3。这是关键，上清收获后，立即启动清洁，不要让容器表面上的干细胞。 </li><li>集中上清至75毫升，使用Centramate切向流过滤系统。 </li><li>加入500毫升的PBS，再浓缩至75毫升。重复3 addit样品的成分，完全缓冲交换。 </li><li>平衡与1×PBS 1毫升抗HA的亲和层析柱和浓缩样品申请由重力流率&lt;1毫升/分钟。 </li><li> 30毫升的1X PBS-Tween20清洗之列。 </li><li>解散医管局肽在1X PBS（1.0毫克/毫升），并于37°C。 </li><li>适用于1毫升医管局肽的抗HA列，并允许肽流入树脂。收集的流量通过。停止流通肽的解决方案时达到床的高度。 </li><li>在37°C孵育15分钟，整个抗HA列。 </li><li>重复步骤12两个额外的时间。 </li><li> 1毫升的1X PBS适用于抗HA列和流入树脂床，直到它到达的高度。收集的流量通过。 </li><li>再生与10毫升0.1 M的甘氨酸pH值2.2的抗HA列。在4°C的0.02％（W / V）在PBS南3 10毫升PBS和存储亲和层析柱洗净。 </li><li>进行SDS-PAGE分析和游泳池的Fractions相应。注:医管局肽与蛋白质浓度测量干涉一个280或布拉德福德。要估计到SDS-PAGE胶蛋白目前，负载5，10，15，25微克的BSA作为标准和比较带强度。 </li></ol>可重复步骤9-17捕捉附加条件培养基蛋白。 4。代表结果在这篇文章中，我们描述和展示毫克，随后可以使用结构和功能研究人类蛋白质的生产数量为方便表达平台。人类蛋白的筛选构建HEK 293T细胞在6孔板的使用效率和有效地确定适合较大规模的生产结构。商业的表达载体可以有效地转染的HEK 293T使用各种转染试剂的细胞，如GeneJuice，FuGene HD或PE，<html>我们建议商业转染试剂的使用，如GeneJuice或FuGene HD，用于测试表达式，这些试剂是穷表达的蛋白（ 图3）更有效。较大规模的表达选择结构的特点应当由一个单一的，强度强带，相应的印迹（ 图3）适当的分子量。糖蛋白，可作为一个更广泛的频段，由于糖基化的异质性迁移。我们已经证明，各种大分子，从病毒的糖蛋白，细胞因子，细胞因子受体，与其他表面蛋白，可以表达和纯化产生的蛋白质的millgram数量使用这种一般的表达平台（ 图4）。 <img alt="图1" src="/files/ftp_upload/4041/4041fig1.jpg" /> 图1。小规模转染的流程示意图 。tp_upload/4041/4041fig1large.jpg"目标="_blank"&gt;点击这里查看大图。 <img alt="图2" src="/files/ftp_upload/4041/4041fig2.jpg" /> 图2。康宁10层为较大规模的蛋白表达CellSTACK。每一层都包含636厘米2细胞附着的表面积。一个标准的实验室二氧化碳培养箱（6.0立方英尺）将舒适地容纳4个10层的细胞工厂。 <img alt="图3" src="/files/ftp_upload/4041/4041fig3.jpg" /> 图3小规模的各种分泌蛋白的表达，我们进行了一系列小规模的测试使用常见的转染试剂的表达:GeneJuice，FuGene HD和爱德华王子岛（一）选定的人类细胞蛋白质的免疫印迹筛选（tetherin），受体。 （IL-2受体的β亚基）和细胞因子（IL-2）。 tetherin是人类的限制释放的膜糖蛋白新生的HIV-1病毒颗粒16。作为糖化，二硫键相连的二聚体〜36 kDa的存在的tetherin外域。还原条件下，如下所示，tetherin迁移作为一个单体〜22 kDa的表观分子量。白细胞介素-2（IL-2）是一种细胞因子（〜17 kDa的）参与淋巴细胞增殖17。它与IL-2受体复杂交互，其中IL-2Rβsubunit（〜26 kDa的）是一个组件18。 CD21的参与补体系统的激活和成熟B细胞是一种膜蛋白，也是EB病毒的受体。糖化CD21的细胞外域迁移作为一个单体〜20 kDa的表观分子量。（二）西方选择表面（XMRV的环境保护和ebolavirus GP）病毒糖蛋白印迹筛选。 XMRV和ebolavirus糖蛋白（跨膜锚删除）三聚尖峰存在于病毒膜，并在参与宿主细胞附着和融合。 XMRV的环境保护和EBOV GP的胞外大量的装饰与N-连接聚糖和迁移的表观分子量为70 kDa和75 kDa的分别。 <img alt="图4" src="/files/ftp_upload/4041/4041fig4.jpg" /> 图4。人体细胞蛋白质纯化大规模的HEK 293T文化 。使用的是10层的细胞工厂所有的蛋白表达，集中和抗HA色谱纯化。考马斯染色的SDS-PAGE分析表明，外域的白细胞介素-2受体（IL-2R）α和γ亚基迁移分子量为40 kDa和46 kDa的分别。的tetherin外域迁移作为一个二聚体，在非还原条件​​下，有一个明显的分子量为36 kDa的。请注意，有一个明显的分子量为60 kDa的出现，在一些BSA的污染。此外，异质性的N-连接聚糖提出tetherin扩大IL-2R的α和IL-2Rγ原因波段的SDS-PAGE凝胶上。这些复杂型N-连接聚糖肽:N-糖苷酶可以去除楼<table border="1"><tbody><tr><td> 船只 </td><td> 表面积 </td></tr><tr><td> 6孔板</td><td> 9.5厘米2（每口井） </td></tr><tr><td> 100 mm培养皿</td><td> 55厘米2 </td></tr><tr><td> 245 mm培养皿</td><td> 500厘米2 </td></tr><tr><td>的T75厘米2瓶</td><td> 75厘米2 </td></tr><tr><td> T175厘米2瓶</td><td> 175厘米2 </td></tr><tr><td> T225厘米2瓶</td><td> 225厘米2 </td></tr><tr><td>滚瓶的定期</td><td> 850厘米2 </td></tr><tr><td>扩大表面滚瓶</td><td> 1700厘米2 </td></tr><tr><td> 1层CellSTACK的</td><td> 636厘米2 </td></tr><tr><td> 2层CellSTACK的</td><td> 1272厘米2 </td></tr><tr><td> 5层CellSTACK的</td><td> 3180厘米2 </td></tr><tr><td> 10层CellSTACK， </td><td> 6360厘米2 </td></tr><tr><td> 40层CellSTACK的</td><td> 25440厘米2 </td></tr></tbody></table>表1。比较细胞培养用于蛋白表达的船只。 试剂配方名单 100X环丙沙星 10毫升溶液，加入10毫升去离子水至10毫克的环丙沙星。加入10微升6N盐酸环丙沙​​星完全溶解。 PEI（1毫克/毫升）为100毫升溶液，溶解在去离子水和热量的25 kDa的线性PEI 100毫克至80°C。冷却溶液至室温，调节pH值至7.2，0.22微米过滤消毒，分装和冻结在-20°为LONÇG-长期储存。 1X PBS洗 1升水溶液:8.0 g氯化钠，氯化钾0.2克，1.4克的Na 2 HPO 4（无水），0.24克KH 2 PO 4。溶液的pH值调整到7.4，并填写到1.0 L。 8.0 g氯化钠，氯化钾0.2克，1.4克的Na 2 HPO 4（无水），0.24克KH 2 PO 4计 ，1毫升吐温20:1X 1升水溶液PBS-吐温20。溶液的pH值调整到7.4，并填写到1.0 L。 Tris碱3.0克，14.4 g甘氨酸，150毫升甲醇:1X 1升水溶液传输缓冲区 。 1X SDS-PAGE电泳运行 1升水溶液缓冲 :3.0克Tris碱，14.4 g甘氨酸，1.0克SDS。 减少 10毫升溶液的SDS-PAGE样品缓冲液 :0.6克SDS，1毫克溴酚蓝，1.8毫升1.0的Tris-盐酸pH值6.8，3毫升甘油，5％（V / V）2 -巯基乙醇。 <p类="jove_content"的框1。细胞繁殖的总协议 <ol><li>杜尔贝科的改良Eagle在5％CO 2饱和湿度在37°C，10％（V / V）胎牛血清（FBS）1X笔/链球菌培养基（DMEM培养基）生长在HEK 293T细胞。 </li><li>倒置显微镜下观察细胞。当细胞在100％汇合，删除和丢弃培养基。 </li><li> 5毫升血清中删除的痕迹无菌的1×PBS冲洗细胞。倒掉PBS洗涤。 </li><li>加入2毫升0.05％（W / V）胰蛋白酶-EDTA溶液1 T225厘米2瓶（或1毫升0.05％（W / V）的T75厘米2瓶胰蛋白酶EDTA），室温孵育直到细胞分离从表面。它也有可能使用无菌1X PBS - EDTA分离​​HEK 293T细胞。 </li><li> 10％（V / V）FBS的DMEM培养液13毫升加入1 T225厘米2瓶（或9毫升培养液，10％（V / V）胎牛血清的T75厘米2瓶），以抑制胰蛋白酶的反应。 </li><li>分裂ÇELLS 1:5。对于T225厘米2瓶，加入1X笔/链球菌和10％（V / V）FBS 3毫升至27毫升的新鲜培养液悬浮细胞，在一艘新的文化。为的T75厘米2瓶，加2毫升细胞悬液至8毫升的新鲜培养基。在5％CO 2饱和湿度在37°C孵育文化。细胞应该在两天内增长到100％汇合。 </li></ol> 专栏2。 Western blot分析 <ol><li>加入10μL的SDS-PAGE减少样品缓冲液30μl的细胞培养上清。负载样品和预染到分子量聚丙烯酰胺凝胶电泳，175 V或1小时，直到分子量标记和使用1X SDS-PAGE电泳运行缓冲electrophorese标记是很好的解决。 </li><li>在100％的甲醇中浸泡1分钟激活PVDF的Immobilon-P膜。 </li><li>组装印迹器具。确保PVDF膜面临的正极和保持湿1X传输缓冲区的所有组件。 AVO聚丙烯酰胺凝胶和膜之间的id气泡。 </li><li>完全填补1X传输缓冲区和1个小时的转移电泳室，在100 V。 </li><li>座1X PBS-Tween20 5％（W / V）脱脂牛奶膜，在室温下1小时或隔夜在4°C。 </li><li>孵化用单克隆抗体溶解于5％（W / V）脱脂牛奶室温1小时或隔夜1X PBS-Tween20（即1:1000稀释的抗HA抗体或其他适当的抗体）在4°C。 </li><li> 10分钟，在1X PBS-Tween20洗膜。重复两个额外的时间。 </li><li>孵育中学单克隆抗体在室温下1小时碱性磷酸酶（1:1000稀释于5％（W / V）脱脂牛奶1X PBS-Tween20）共轭，或隔夜在4°C。 </li><li> 10分钟，在1X PBS-Tween20洗膜。重复两个额外的时间。 </li><li>膜放入一个小容器，添加5毫升碱性磷酸酶substraTE（BCIP / NBT）的解决方案。彩色的发展应在1-5分钟内发生。一旦所需的乐队强度，达到清洗，去离子水和空气干燥。颜色随着时间的推移可能会褪色;电子扫描一次干的膜。 </li></ol> 专栏3。清洗和回收利用细胞培养容器 虽然电池的工厂被设计为一次性使用，这些船只可以回收额外的大规模使用下列清洁协议的转染: <ol><li>从10层的细胞工厂的调迁上清后，立即加入20％（V / V）的漂白剂和大力摇晃来分离细胞。在室温下孵育了三个小时。 </li><li>清空容器，并加入新鲜的20％（V / V），漂白剂在室温下过夜孵育。 </li><li>清空船只，并用1.5去离子水的大号。重复三次。 </li><li>清空10层的细胞工厂，并添加0.5无菌1×PBS大号辅以10X抗生素/抗霉菌的解决方案。储存在室温下的船只和更换灌装之前下使用的端口（标准的33毫米螺纹帽）排气帽。注:所有层应清洗后完全清楚，如果没有，不使用和处置电池厂根据机构的指导方针。 </li></ol>

<ol>
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没有利益冲突的声明。

10层的细胞工厂生产毫克的蛋白质数量的有效容器。使用超过其他传统的船只，如滚瓶，摇瓶或微调瓶，电池工厂的一个主要优点是，它们不需要任何额外的实验室设备的采购。一个标准的二氧化碳培养箱（6.0立方英尺）轻松容纳4个10层的细胞工厂（ 图2）。此外，这些船只需要较少的劳动比菜，瓶或滚瓶细胞和蛋白质的产生和空间;一个10层的细胞工厂，相当于使用7.5定期滚瓶（ 见表1）。更重要的是，很好地适应HEK 293T细胞中的船只，非常适合由PEI瞬时转染的，从而提供了一种低成本和有效的选择商业的转染试剂。虽然电池的工厂被设计为一次性细胞培养塑料制品，我们有bEEN能够有效地清理和重新使用这些船只多次，没有污染问题，从而大大降低其每次使用成本。瞬态蛋白表达，使用的是10层的细胞工厂能够生产〜1-10毫克纯化蛋白（ 图4），根据蛋白质和其修改。这是可以去克隆和小规模表达大规模表达和纯化约3-4周完成。总之，这是一种快速，一般的平台，是非常有用的人或病毒表面的蛋白质分泌的生产。此外，这个平台也可以被用来表达抗体，病毒样颗粒，腺病毒和慢病毒基因转移。 下面我们列出了常见的问题和潜在的解决方案。更详细的故障排除指南，请参阅14。 <ol class="lalpha"><li> 没有或非常低的蛋白表达: <ul><li>标准普尔Ĥealth的细胞支原体污染的可行性和试验使用台盼蓝染色细胞。如果细胞有高通道数（&gt; 25代），增长缓慢，开始新的HEK 293T细胞的新鲜。由年龄在培养的细胞转染效率和蛋白生产受到影响。 </li><li>应差的瞬时转染细胞转染汇合在40％的水平。应该优化的DNA转染试剂的比例。 </li><li>蛋白质是不稳定或不折叠检查不溶性蛋白质的细胞沉淀。测试其他结构或同源蛋白的表达。 </li></ul></li><li> 没有蛋白质被洗脱列: <ul><li>蛋白质保留0.1 M甘氨酸pH值2.2上柱洗脱感兴趣的蛋白质印迹分析。如果保留蛋白，洗脱的合成医管局肽高浓度使用，或在37°C孵育较长时间的列。 </li><li>反......上校olumn被损坏而列，如果在适当的清洗和存储，可多次重复使用，列的效率将随时间而减少。使用一种新的抗HA列。 </li><li>蛋白质的降解，添加蛋白酶抑制剂，在整个净化过程中的蛋白质，并保持在冰上。 </li><li>蛋白沉淀或聚集上的列，如果蛋白质是不稳定的，尝试不同的结构或缓冲区的条件（如pH值，盐，添加剂等），以提高蛋白质的稳定性。 </li><li>蛋白质未能绑定到列医管局标签可能被埋没或无法访问的。检查看到，蛋白质是不是在流或清洗。 </li></ul></li><li> 血清白蛋白的污染: 牛血清白蛋白（BSA），往往非特异性结合的抗HA树脂和可能污染医管局肽洗脱。图。 4，牛血清白蛋白作为一个紧凑〜60-65 kDa的表观分子量带迁移。在这种情况下，Tween-20的浓度增加在1X带够BS-Tween20洗到0.2％（V / V）和洗量增加。另外，BSA的污染，使用大小排斥或离子交换层析，可能会被删除。 </li></ol>

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td>评论</td></tr><tr><td>碱性磷酸酶（BCIP / NBT）的液体底物溶液。 </td><td>西格玛</td><td> B6404 </td><td></td></tr><tr><td>抗生素/抗霉菌，100X </td><td> Invitrogen公司</td><td> 15240062 </td><td></td></tr><tr><td>抗HA的亲和力矩阵，克隆3F10 </td><td>罗氏公司</td><td> 1815016 </td><td></td></tr><tr><td>鼠单克隆抗体，抗-HA克隆16B12 </td><td> Covance公司</td><td> MMS-101P </td><td></td></tr><tr><td>细胞培养瓶的T75厘米2组织文化对待</td><td>康宁</td><td> 430641 </td><td></td></tr><tr><td>细胞培养瓶，T225厘米2组织文化对待</td><td>康宁</td><td> 431082 </td><td></td></tr><tr><td>细胞培养板,6-以及组织培养处理</td><td>康宁</td><td> 3516 </td><td></td></tr><tr><td>电池厂，10层CellSTACK的; </td><td>康宁</td><td> 3312 </td><td></td></tr><tr><td> centramate欧米茄5K盒式</td><td>鲍尔</td><td> OS005C12 </td><td></td></tr><tr><td> centramate欧米茄30K磁带</td><td>鲍尔</td><td> OS030C12 </td><td></td></tr><tr><td>玻璃层析柱，1.0×10厘米</td><td> kontes </td><td> 4204001010 </td><td></td></tr><tr><td>环丙沙星</td><td>西格玛</td><td> 17850 </td><td></td></tr><tr><td>的CO 2 </td><td></td><td></td><td></td></tr><tr><td>杜尔贝科的改良Eagle的媒体（DMEM培养基） </td><td>西格玛</td><td> D5796 </td><td></td></tr><tr><td>胎牛血清（FBS），热灭活</td><td> Invitrogen公司</td><td> 12484-028 </td><td></td></tr><tr><td> FuGENE的HD转染试剂</td><td> Promega公司</td><td> 4709691001 </td><td></td></tr><tr><td> GeneJuice转染试剂</td><td> EMD /默克</td><td> 70967-6 </td><td></td></tr><tr><td>甘氨酸</td><td>西格玛</td><td> G8898 </td><td></td></tr><tr><td>山羊抗鼠IgG F（AB&#39;）2碱性</td><td> Thermo Scientific的</td><td> 31324 </td><td></td></tr><tr><td>磷酸酶抗体</td><td></td><td></td><td></td></tr><tr><td>血凝素（HA）肽，100毫克</td><td>金斯瑞</td><td>定制合成</td><td></td></tr><tr><td> （序列YPYDVPDYA纯度为95％） </td><td></td><td></td><td></td></tr><tr><td> HEK 293T细胞</td><td> ATCC </td><td> CRL-11268 </td><td></td></tr><tr><td> householð漂白剂（4％W / V次氯酸钠） </td><td>各种品牌</td><td></td><td></td></tr><tr><td>的Immobilon - P PVDF膜</td><td> Millipore公司</td><td> IPVH07850 </td><td></td></tr><tr><td>小量质粒纯化试剂盒，快速PureLink </td><td> Invitrogen公司</td><td> K2100-11 </td><td></td></tr><tr><td> MaxiPrep质粒纯化试剂盒，PureLink HiPure </td><td> Invitrogen公司</td><td> K2100-07 </td><td></td></tr><tr><td>南三 </td><td>西格玛</td><td> S8032 </td><td></td></tr><tr><td> pDISPLAY表达载体; </td><td> Invitrogen公司</td><td> V660-20 </td><td></td></tr><tr><td>青霉素/链霉素（笔/链球菌），100X </td><td> Invitrogen公司</td><td> 15140-122 </td><td></td></tr><tr><td>磷酸盐缓冲液（PBS），无菌1X </td><td>西格玛</td><td> D8537 </td><td></td></tr><tr><td>聚乙烯亚胺（PEI），线性25 kDa的</td><td> polyscience </td><td> 23966 </td><td></td></tr><tr><td>脱脂牛奶干粉</td><td>康乃馨</td><td></td><td></td></tr><tr><td> stericup-GP PES真空过滤装置， </td><td> Millipore公司</td><td> SCGPU05RE </td><td></td></tr><tr><td> 0.22微米，500毫升容量</td><td></td><td></td><td></td></tr><tr><td>台盼蓝</td><td> Invitrogen公司</td><td> 15250061 </td><td></td></tr><tr><td>胰蛋白酶-EDTA，0.05％（W / V） </td><td> Invitrogen公司</td><td> 25300-054 </td><td></td></tr><tr><td>吐温20 </td><td>西格玛</td><td> P7949 </td><td></td></tr><tr><td>丙戊酸</td><td>西格玛</td><td> P4543 </td><td></td></tr><tr><td> Centramate切向流系统</td><td>鲍尔</td><td></td><td></td></tr><tr><td> CO 2培养箱孵化器，标准的6.0立方米。英尺。 </td><td>各种品牌</td><td></td><td></td></tr><tr><td>电泳和转印装置</td><td>各种品牌</td><td></td><td></td></tr><tr><td>孵化器，37℃ </td><td>各种品牌</td><td></td><td></td></tr></tbody></table>

a convenient and general expression platform for the production of secreted proteins from human cells

重组蛋白在细菌中表达，通常大肠杆菌大肠杆菌 ，一直毫克的蛋白质数量表达的最成功的战略。然而，原核主机通常是不为人类，病毒或真核蛋白质由于外国高分子毒性，在蛋白质折叠机制的差异，或因缺乏特别合作或翻译后的修改，在细菌的适当表达。基于酵母（ 毕赤酵母或酵母 ）1,2，杆状病毒感染的昆虫（S.秋行军虫或 T 妮 ）细胞3，和无细胞体外翻译系统的表达系统2,4已被成功地用于生产哺乳动物蛋白质。直观，最匹配的是，使用哺乳动物的主机，以确保包含正确的翻译后修饰的重组蛋白的生产。一些哺乳动物细胞株人类胚胎小子（滋肾（HEK）293，C V-1细胞在 O rigin携带的 S V40 larget T抗原（COS）中国仓鼠卵巢（CHO），和其他人）已成功地利用过度毫克数量的人体蛋白质数量5-9。然而，使用哺乳动物细胞中的优势往往成本较高，专业实验室设备的需求，降低蛋白质产量，和漫长的时间发展稳定表达细胞株的反击。提高产量和生产蛋白质的速度，同时保持成本低，是许多学术和商业实验室的主要因素。 在这里，我们描述了时间和成本效益，由两部分组成，附着HEK 293T细胞分泌的人类蛋白质表达过程。这个系统是能够生产微克到毫克数量的功能蛋白质结构，生物物理和生物化学研究。第一部分，是产生多个感兴趣的基因结构并行和瞬时转成小规模的附着的HEK 293T细胞。重组蛋白分泌到细胞培养液中的检测和分析，进行印迹分析，使用市售的抗体对矢量编码的蛋白质纯化标签指示。随后，适合大规模蛋白质生产结构瞬时转染使用聚乙烯亚胺（PEI）在10层的细胞工厂。成升体积的条件培养基中分泌蛋白集中到管理大量使用切向流过滤，净化，抗HA的亲和层析。这个平台的效用证明毫克量的细胞因子，细胞因子受体，细胞表面的受体，内在的制约因素，与病毒糖蛋白的表达能力。这种方法也被成功地用于三聚ebolavirus糖5,10结构的决心。 培养箱 ，是必需的。这个过程可能会迅速扩大到更复杂的系统，如蛋白质复合物，抗原和抗体，疫苗生产的病毒样颗粒，或生产困难的细胞株转导的腺病毒或慢病毒表达。

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

在人类后基因组时代，在本机构的重组蛋白的结构，功能和治疗的研究和发展是至关重要的。在这里，我们描述了测试和大规模的蛋白质在人类胚胎肾细​​胞293T，可用于生产各种重组蛋白表达系统。

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. ...

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JoVE Journal

Biology

21.1K 次观看.  University of Toronto. 在人类后基因组时代，在本机构的重组蛋白的结构，功能和治疗的研究和发展是至关重要的。在这里，我们描述了测试和大规模的蛋白质在人类胚胎肾细​​胞293T，可用于生产各种重组蛋白表达系统。

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

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

人类胚胎干细胞（ES细胞）的繁殖

科研

生物学

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

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 Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

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 ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

高级别瞬态表达重组蛋白的植物高效的农杆菌

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

高效的生产和纯化重组小鼠Kindlin-3从昆虫细胞生物物理研究

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

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 ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

从富集组织及消化细胞外基质蛋白的成肽质谱分析

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

一个从人体细胞分泌蛋白生产的方便，一般的表达平台

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