Name: comparison of tobacco host cell protein removal methods by blanching intact plants or by heat treatment of extracts
Uploaded: 2016-08-08T15:00:00.000+00:00
Duration: 11 min 6 s
Description: Watch this Scientific Journal Video about Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts at JoVE.com

We would like to acknowledge Dr. Thomas Rademacher, Alexander Boes and Veronique Bei&#223; for providing the transgenic tobacco seeds, and Ibrahim Al Amedi for cultivating the tobacco plants. The authors wish to thank Dr. Richard M. Twyman for editorial assistance as well as G&#252;ven Edg&#252; for providing the MSP1-19 reference. This work was funded in part by the European Research Council Advanced Grant ''Future-Pharma'', proposal number 269110, the Fraunhofer-Zukunftsstiftung (Fraunhofer Future Foundation) and Fraunhofer-Gesellschaft Internal Programs under Grant No. Attract 125-600164.

1.培育烟草的研究<ol><li>冲洗的去离子水1〜2升，随后每矿棉块用1L 0.1％[w / v的]肥料溶液。将在每个矿棉块的一个烟草种子和0.25升的肥料溶液冲洗轻轻没有洗去种子16。 </li><li>培育7周的烟草植株中，用70％相对湿度的温室中，16小时的光周期（180微摩尔仲丁基- 1米 - 2;λ= 400 - 700 nm），而一个25/22℃的光照/黑暗温度制度。 </li><li>收获所有树叶以外的四个子叶叶子，分别位于在植物茎的基部。 </li></ol> 2.可选:热沉淀通过热烫注:执行，以便通过热烫沉淀烟草医疗专业人员在步骤2.1中所述2.12的步骤。跳过整个部分2，如果医疗专业人员将沉淀在加热的容器（部分4）或使用热交换器（部分5）。 <ol><li>抛开50克植物材料并进行提取不热烫（第3节）。以这种提取物的样本为后续分析（第7条）过程中的内部控制。 </li><li>设置一个8-L工作体积水浴， 例如 ，50×40×40厘米，在绝热发泡聚苯乙烯桶或类似。安装在水浴用于随后的温度控制（步骤2.7）的可调节的恒温器。 </li><li>整个组件转移到磁性搅拌盘，并放置在水浴磁力搅拌棒。确保磁场足够强的旋转搅拌棒。 </li><li>至少有四个支撑砖在其聚丙烯篮（步骤2.6）将被漂白过程中放置​​后环绕搅拌棒。确保支承瓦片由非磁性材料（ 例如 ，不锈钢合金含有镍）制成，它们比搅拌棒（ 图2）更高。 </li><li> 8升的去离子水加入到水浴中。 注意:使用的缓冲液， 例如 ，50mM磷酸钠（pH7.5）中，可以提高靶蛋白的产量，如果低pH沉淀是一 ​​个问题。 </li><li>放置一个23×23×23 5cm聚丙烯篮子在支撑瓷砖和确保篮不与搅拌棒旋转干扰，并且它是在液体中完全浸没。如果有必要，添加额外的水/缓冲，直到篮完全浸入，然后再取出篮筐。 注意:所有的后续步骤高达2.12涉及热液体的处理。包括佩戴隔热手套适当的个人防护装备。 </li><li>使用可调节的恒温器以使水浴至70℃（或用于实验所需的温度）。等待至少15分钟达到后所要求的温度，以确保整个组件已经达到热表面升平衡。 </li><li>准备从收获的烟草簧片150克一份。将一个等份在篮下，同时避免不可逆压缩和叶片损伤， 例如，通过撕裂。避免过量填充以植物材料或后者的致密填料篮筐。 </li><li>小心，但很快淹没在篮下的热的液体，并将其放置在支撑瓷砖。放置在篮下，以防止漂浮顶部的不锈钢块。 </li><li>孵育5分钟的叶子在漂白液，或选择时间相适应的实验设计。显示器在整个孵化期液体温度。 </li><li>小心地从漂白液篮筐，让从叶子残留的液体排出，持续30秒。然后把植物的叶子筐而出，他们转移到搅拌机并立即开始提取（第3节）。 注:植物可以热烫后和第开始前保持延长的时间段ê提取， 例如，在冰上超过30分钟或在-20℃冷冻几个星期已经成功地测试。然而，产品稳定性可能随储存时间，因此建议立即处理下滑。 </li><li>直到整个收获的生物质进行处理，重复步骤2.8至2.11与新鲜叶材料。 </li></ol> 3.从烟叶中提取的蛋白质注意:以下步骤涉及与旋转叶片搅拌机。而它安装在搅拌器马达在搅拌器桶不工作。 <ol><li>放置收获（步骤1.3）或热烫（步骤2.11）150克（湿质量）叶在搅拌器和添加450毫升提取缓冲液（50mM磷酸钠，500mM的氯化钠，10mM的亚硫酸氢钠，pH值8.0）。 注意:提取缓冲液组成取决于要提取的蛋白上，并因此可能需要调整， 例如 ，另一个pH值的使用或缓冲组分，如TR是。 </li><li> 30秒穿插场所均质叶片3×30秒脉冲。确保叶子混匀后，不堵塞果汁桶。停止搅拌器并提起叶如有必要，以防止堵塞，然后继续同质化。 </li><li>就拿这对于后续的分析（第7条）生产的每提取1毫升样品。如果该植物材料烫，继续部分6。否则，在一个容器中热沉淀（第4节）或热交换器（部分5）继续，这取决于所选择的实验方法。 </li></ol> 4.可选:热火降水搅拌槽内注:进行，以便在搅拌容器沉淀烟草医疗专业人员在4.2节到4.11描述的步骤。跳过整个部分4中，如果医疗专业人员已热烫（第2部分）中沉淀或将使用热交换器（第5部分）中沉淀。 <ol><li>设置了两个8-L工作体积的水浴， 例如，50×40×40厘米，在热绝缘聚苯乙烯泡沫塑料或类似。在第一次洗澡，挂载为后续的温度控制（步骤4.6）的可调恒温。在第二个中，添加5升去离子水和2kg冰随后的冷却（步骤4.9）的。 </li><li>传送所述第一水浴上磁性搅拌板，以及2升不锈钢容器中放入水浴，使得容器的中心对准到搅拌板的中心。 </li><li>放置在不锈钢容器中的磁性搅拌棒。确保磁场足够强的旋转搅拌棒。 </li><li>填充去离子水的水浴中以5厘米的不锈钢容器的上边缘的下方。然后填充提取缓冲液的容器中。放置在容器的聚苯乙烯泡沫盖。 注意:所有的后续步骤高达4.9涉及热液体的处理。包括穿热插件适当的个人防护装备ulated手套。 </li><li>通过在聚苯乙烯泡沫盖的西装料孔插入温度计入不锈钢容器中。设置水浴温度至78℃，并培育整个组件15分钟以达到热平衡。保证在容器中的温度为70℃，约8℃下的水浴的设定点以下。 </li><li>如果在不锈钢容器中的温度从70℃不同，相应地调整水浴的温度，并让系统平衡另一个15分钟。重复此步骤直到在容器中的温度为70℃或根据需要进行实验，并清空不锈钢容器中。 </li><li>倾300毫升提取物（步骤3.2）的入不锈钢容器中，而它仍然在水浴并启动计时器。搅拌该提取物在150rpm和孵育5分钟。确保了提取在此潜伏期达到70℃至少2分钟的温度。 </li><li>除去从磁性搅拌器热水浴，取出不锈钢容器中，并将其放置在冰冷的桶。放置后者上磁力搅拌器和除去聚苯乙烯泡沫盖。 </li><li>确保植物匀浆以150rpm充分搅拌，放置温度计提取物中孵育，直到达到20℃的温度下或通过实验设计规定的温度。 </li><li>重复步骤所有等分4.7至4.9。取每个热的1ml样品沉淀提取一旦达到最终温度，然后与第6条进行。 </li></ol> 5.可选:热火降水热交换器注:进行以沉淀用热交换器烟草医疗专业人员在节5.2至5.12所述的步骤。跳过整节5，如果医疗专业人员已被漂白（第2节），或在加热容器（第4节）沉淀。 <ol><li>设置了两个8-L工作体积水浴， 例如，50×40×40厘米，在热绝缘聚苯乙烯泡沫塑料桶或相似。在第一次洗澡，挂载为后续的温度控制（步骤5.3）的可调恒温。在第二个中，添加5升去离子水和2kg冰随后的冷却（步骤5.10）的。 注意:所有的后续步骤高达5.9涉及热液体的处理。包括佩戴隔热手套适当的个人防护装备。 </li><li>填用8L去离子水的第一水浴中。使用温控器温度设定为74.5℃。孵育组装15分钟才能达到热平衡。 </li><li>通过将0.5升塑料烧杯到1升塑料烧杯制备绝缘储存容器和填充用脱脂棉的空白。或者，使用专用的隔热容器用0.5升工作体积。 </li><li>热交换器连接到蠕动泵在一端，并在水净化的出口软管- [R结束使用L / S 24管子。放置两管道在隔热存储容器温度计端沿着并用300ml提取缓冲液（ 图2）填充。 </li><li>放置热交换器进入热水浴中，以300ml分钟-1的速度启动蠕动泵。保证在容器中产生的温度为70℃，水浴的设定点低于约4.5℃，3分钟后， </li><li>如果在绝缘容器中的温度由70℃的不同，相应地调整水浴的温度，并让系统平衡另外15分钟重复此步骤直至从环境提取缓冲液的温度上升至70℃，在不到3分钟，或等于该所需的实验，并清空不锈钢容器中。 </li><li>从绝热容器丢弃提取缓冲液和制备的植物提取物的300ml等分试样。一个等分填充绝热容器。 </li><li>泵通过热交换器的植物提取物（第3.3节）以300ml分钟-1 5分钟。确保该萃取温度为70℃3分钟后，或等于在实验设计中定义的温度。 </li><li> 5分钟后，将热交换器入冰冷水浴而提取仍在泵送。孵育在此设置，直到温度达到正好是20℃，或在实验设计中定义的温度。 </li><li>移除连接到从绝缘容器中的蠕动泵的入口软管和继续泵送以从热交换器内收集余热沉淀植物提取物。然后停泵。 </li><li>重复步骤所有等分5.7至5.10。每次取热沉淀提取物1毫升样品一旦达到最终温度，然后通过对提取物的袋式过滤（第6条）。 注意:在第一循环通过步骤5.7至5.10将没有提取缓冲液对丢弃在步骤5.7。 </li></ol> 6.植物提取物的袋式除尘<ol><li>装入袋滤器成装配到过滤器壳体，并提供了灵活的过滤材料的机械支撑的相应支撑篮。放置1升容器中的篮的下方，并在150毫升分钟-1的速率应用提取等分试样（3.3节，4.10或5.11，这取决于所选热处理）到袋。 </li><li>过滤后，用浊度计测量1:10稀释滤液的浊度在提取缓冲液。 </li><li>取1毫升样品和处理袋滤液作为在实验设计中定义， 例如，深度过滤和层析7,10。 </li></ol> 7.样品分析<ol><li>测量总可溶性蛋白（TSP）的使用Bradford方法24,25的数量。 <ol><li>一式三份，吸取2.5微升各样品成一个单一的孔96孔板中。包括一式三份8牛血清白蛋白（BSA）标准覆盖范围0 - 2000微克毫升-1。 </li><li>加入200μlBradford试剂至各孔，并通过上下抽吸调匀而是轻轻足以避免形成气泡。 </li><li>孵育10分钟，在22℃下并测量在分光光度计在595nm处的吸光度。计算基于通过BSA参考点的标准曲线对样品中的TSP浓度。 </li></ol></li><li>通过表面等离子体共振光谱26量化Vax8。 <ol><li>设置的表面等离子体共振仪器与4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES）运行缓冲液（10mM HEPES，3mM的乙二胺四乙酸（EDTA），1500 5mM氯化钠，0.05％体积/体积的聚山梨醇酯20， pH为7.4），并装入羧甲基葡聚糖表面芯片到设备中。使用的主要功能冲洗系统和启动手动运行具有30的流速微升分钟-1以上流动池1和用25mM氢氧化钠60秒穿插注射2注入的30mM氯化氢60秒两次。 </li><li>制备200微升的500微克毫升-1单克隆抗体5.2的10mM乙酸钠（pH 4.0）溶液。解冻1-乙基-3-（3-二甲基氨基丙基）碳二亚胺盐酸盐（EDC）和N-羟基琥珀酰亚胺（NHS）的小瓶中，并以16,000 xg离心离心他们1分钟。混合70微升的EDC用70微升的NHS的和传送到7毫米的塑料小瓶中。 </li><li>通过以10微升分钟-1的流速注入10分钟的EDC / NHS混合物在流动池1和2活化羧甲基化的葡聚糖表面芯片表面。 </li><li>设置流仅小区2的流动路径。夫妇单抗5.2通过在10微升分钟-1的速度注入它为15分钟。 </li><li>切换流路中流动单元1和2，并在5微升分钟-1停用的表面的速率注入乙醇胺7分钟。 ŧ母鸡注入30mM的氯化氢两次60秒用25mM氢氧化钠60秒穿插注射。 </li><li>注入样品和MSP1 - 19标准，在30微升min的速率为 180秒。确保Vax8浓度为50 - 1000毫升纳克-1。制备预稀释液在HEPES缓冲的含有EDTA和聚山梨醇酯20（HBSEP）如有必要，盐水。 </li><li>从流通池2中减去流通池1中的峰值信号，并使用差来计算基于所述MSP 1的信号在采样Vax8浓度- 19注射用500纳克毫升-1的已知蛋白质浓度。 </li><li>再生各样品或MSP 1后的芯片表面- 19喷射通过暴露于30mM的氯化氢60秒在30微升min的流速-1。 </li></ol></li><li>通过量化荧光光谱红色荧光蛋白<ol><li>一式三份，吸管将50μl各样品的成黑色半芳的单个孔EA 96孔板。包括六个红色荧光蛋白标准覆盖范围0 - 225微克毫升-1。 </li><li>使用530±30nm的激发滤光器和分光光度计590±35 nm的发射滤光器测量一式两份的荧光。计算基于通过DsRed的参考点的标准曲线对样品中的红色荧光蛋白浓度。 </li></ol></li><li>粒度分布的测定<ol><li>洗试管用1毫升异丙醇中，然后用2ml的去离子水以去除灰尘粒子。吸管850微升样品放入试管。 </li><li>放置反应杯插入ζ电位和粒度分析仪。打开"软件"，并开始与"蛋白质"的物质和"PBS"作为分散剂手动测量。选择25℃的温度下和180秒的平衡时间。 </li><li>选择在173°的反向散射"DTS0012"细胞与措施。选中"自动"FUNCT离子的测量持续时间和选择三个测量与没有散布的延迟。 </li><li>调查粒度分布峰轮廓一次测量是通过选择在实验视图样品的三次测量完成。选择"音量PSD"标签。 </li></ol></li></ol>

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作者有没有利益冲突披露。

以上所述的三种方法为热沉淀能有效地去除前的任何层析纯化步骤16,17烟草医疗专业人员。它们补充，旨在增加初始产品的纯度， 例如，吐水29，rhizosecretion 30或离心萃取31,32，所有这些都限于分泌蛋白的其他策略。但是，基于热的方法只能在一个有意义的方式使用，如果待纯化的目的蛋白可承受〜60℃的最低析出温度超过1分钟。因此，在任何的三种方法的?...

现代医疗保健系统越来越依赖于生物制药蛋白质1。在植物中生产这些蛋白质相比于常规表达系统2-4由于低病原体负担和更大的可扩展性是有利的。然而，植物性药物下游处理（DSP）是很有挑战性的，因为颠覆性的提取程序导致高粒子负担，与浊度超过5000浊度单位（NTUs）和宿主细胞蛋白（HCP）含量往往超过95 ％[M / M] 5,6。 精细澄清过程需要以除去分散颗粒7-9，但色谱仪器是如果存在用于高效医疗专业人员去除10,11较早步骤初始产物回收过程中结合和洗脱模式运行成本更低。这可以通过沉淀使用floccul靶蛋白可以实现任蚂蚁12或低pH 13,14，以及通过使医疗专业人员聚集。核酮糖-1,5-二磷酸羧化酶/加氧酶（Rubisco），最丰富的HCP在绿色植物如烟草（ 烟草 ），所述的选择性汇聚可以通过添加聚乙二醇15来促进，但是这是昂贵且具有大不相容-scale制造。热处理已显示变性和沉淀烟草医疗专业人员的超过95％，而蛋白质疟疾疫苗候选物如Vax8留在溶液中16-18稳定。 三种不同的方法被用来实现烟草医疗专业人员的热诱导析出:（ⅰ）热烫， 即，完整叶片在热液体的浸入，（ⅱ）一个温度受控的搅拌容器，和（iii）的热交换器（ 图1）16。对于完整叶片，漂烫实现医疗专业人员的快速，高效的降水，也容易扩大规模，并与现有的大型的制造工艺，其中包括一个初始步骤洗植物生物质19兼容。与此相反，温度控制的容器已经在一些工艺可用，并且可以用于植物的热处理提取20，但其可扩展性和能量转移率是有限的，因为罐的表面-体积比逐渐减少，并变得不适合于生产规模。的热交换器是一个在技术上明确替代加热搅拌容器，但需要加热和冷却介质， 例如，蒸汽和冷水的供应充足，以及其适合于热交换器的几何形状的严格控制的体积流动速率和媒体属性， 例如 ，比热容量。本文显示了所有三种方法可如何用于在普通烟草医疗专业人员和植物医疗专业人员的热诱导沉淀。建立和EAC的操作在实验室设置h方法可用于评估其对于大规模的工艺的适用性。主要的挑战是确定适当尺度缩小的模型和运行条件类似于过程大规模生产过程中使用的设备和条件的每个操作。此处呈现的数据是指与表达的疟疾疫苗候选Vax8和荧光蛋白DsRed的16转基因烟草植物进行的实验，但该方法也已成功地应用到N.植物本生瞬时表达等生物制药蛋白21。 一个设计-的-实验（DOE）接近22可促进过程发展，絮凝剂23也可以是在这种情况下有益的，如前所述8。热烫，加热的容器和热交换器之间的主要区别是，热烫施加到完整叶片在过程的早期，而OT她的被施加到植物提取物（ 图1）。 <img alt="图1" src="/files/ftp_upload/54343/54343fig1.jpg" /> 图 1: 工艺流程图图解三种不同方法进行烟草HCP热沉淀实施植物材料洗净，澄清和纯化前均化。用于漂白步骤（红色）的设备可以很容易地添加到现有的机械。相比之下，使用搅拌容器（橙色）和特别是换热器（蓝色）需要一个或几个额外的设备和管道。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54343/54343fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<table><tbody><tr><td>2100P Portable Turbidimeter</td><td>Hach</td><td>4650000</td><td>Turbidimeter</td></tr><tr><td>Amine Coupling Kit</td><td>GE Healthcare</td><td>BR100050&amp;nbsp;</td><td>SPR chip coupling kit</td></tr><tr><td>Autoclaving basket</td><td>Nalgene</td><td>6917-0230</td><td>Basket for leaf blanching</td></tr><tr><td>Biacore T200</td><td>GE Healthcare</td><td>28-9750-01</td><td>SPR device</td></tr><tr><td>Bio Cell Analyser BCA 003 R&amp;amp;D with 3D ORM</td><td>Sequip</td><td>n.a.</td><td>Particle size analyzer</td></tr><tr><td>Blender</td><td>Waring</td><td>800EG</td><td>Blender</td></tr><tr><td>BP-410</td><td>Furh</td><td>2632410001</td><td>Bag filter</td></tr><tr><td>Centrifuge 5415D</td><td>Eppendorf</td><td>5424 000.410</td><td>Centrifuge</td></tr><tr><td>Centrifuge tube 15 ml</td><td>Labomedic</td><td>2017106</td><td>Reaction tube</td></tr><tr><td>Centrifuge tube 50 ml self-standing</td><td>Labomedic</td><td>1110504</td><td>Reaction tube</td></tr><tr><td>CM5 chip</td><td>GE Healthcare</td><td>BR100012&amp;nbsp;</td><td>Chip for SPR measurements</td></tr><tr><td>Cuvette 10 x 10 x 45</td><td>Sarsted</td><td>67.754</td><td>Cuvette for Zetasizer Nano ZS</td></tr><tr><td>Design-Expert(R) 8</td><td>Stat-Ease, Inc.</td><td>n.a.</td><td>DoE software</td></tr><tr><td>Disodium phosphate</td><td>Carl Roth GmbH</td><td>&amp;nbsp;4984.3&amp;nbsp;</td><td>Media component</td></tr><tr><td>Ferty 2 Mega</td><td>Kammlott</td><td>5.220072</td><td>Fertilizer</td></tr><tr><td>Forma -86C ULT freezer</td><td>ThermoFisher</td><td>88400</td><td>Freezer</td></tr><tr><td>Greenhouse</td><td>n.a.</td><td>n.a.</td><td>For plant cultivation</td></tr><tr><td>Grodan Rockwool Cubes 10 x 10 cm</td><td>Grodan</td><td>102446</td><td>Rockwool block</td></tr><tr><td>Twentey-loop heat exchanger (4.8 m length)</td><td>n.a. (custom design)</td><td>n.a.</td><td>Heat exchanger</td></tr><tr><td>HEPES</td><td>Carl Roth GmbH</td><td>9105.3</td><td>Media component</td></tr><tr><td>K200P 60D</td><td>Pall</td><td>5302303</td><td>Depth filter layer</td></tr><tr><td>KS50P 60D</td><td>Pall</td><td>B12486</td><td>Depth filter layer</td></tr><tr><td>Lauda E300</td><td>Lauda Dr Wobser GmbH</td><td>Z90010</td><td>Water bath thermostat</td></tr><tr><td>L/S 24</td><td>Masterflex</td><td>SN-06508-24</td><td>Tubing</td></tr><tr><td>mAb 5.2</td><td>American Type Culture Collection</td><td>HB-9148</td><td>Vax8 specific antibody</td></tr><tr><td>Masterflex L/S</td><td>Masterflex</td><td>HV-77921-75</td><td>Peristaltic pump</td></tr><tr><td>Miracloth</td><td>Labomedic</td><td>475855-1R</td><td>Filter cloth</td></tr><tr><td>MultiLine Multi 3410 IDS</td><td>WTW</td><td>WTW_2020</td><td>pH meter / conductivity meter</td></tr><tr><td>Osram cool white 36 W</td><td>Osram</td><td>4930440</td><td>Light source</td></tr><tr><td>Phytotron</td><td>Ilka Zell</td><td>n.a.</td><td>For plant cultivation</td></tr><tr><td>Sodium disulfite</td><td>Carl Roth GmbH</td><td>8554.1</td><td>Media component</td></tr><tr><td>Sodium chloride</td><td>Carl Roth GmbH</td><td>P029.2</td><td>Media component</td></tr><tr><td>Stainless-steel vessel; 0.7 kg 2.0 L; height 180 mm; diameter 120 mm</td><td>n.a. (custom design)</td><td>n.a.</td><td>Container for heat precipitation</td></tr><tr><td>Synergy HT</td><td>BioTek</td><td>SIAFRT</td><td>Fluorescence and spectrometric plate reader</td></tr><tr><td>VelaPad 60</td><td>Pall</td><td>VP60G03KNH4</td><td>Filter housing</td></tr><tr><td>Zetasizer Nano ZS</td><td>Malvern</td><td>ZEN3600</td><td>DLS particle size distribution measurement</td></tr><tr><td>Zetasizer Software v7.11</td><td>Malvern</td><td>n.a.</td><td>Software to operate the Zetasizer Nano ZS device</td></tr></tbody></table>

comparison of tobacco host cell protein removal methods by blanching intact plants or by heat treatment of extracts

植物不仅对人类提供食物，饲料和原料，但也已经被开发作为一种经济的生产系统，用于生物制药的蛋白质，如抗体，疫苗候选和酶。这些必须从植物生物质进行纯化，但色谱法步骤是由高浓度的植物提取液中宿主细胞蛋白（医疗专业人员）的阻碍。然而，大多数医疗专业人员不可逆聚合在温度高于60℃，促进靶蛋白的随后的纯化。这里，被呈现给实现烟草医疗专业人员的热沉淀在任一完整叶片或提取物的三种方法。完整叶片的热烫可以容易地纳入现有过程，但可能对随后的过滤步骤产生负面影响。相反的是一种用于在搅拌容器叶片提取物的热析出，从而可以提高下游操作的性能虽然有在工艺设备设计的重大变化，如真均质几何。最后，一​​个热交换器设置是公特征的传热条件方面和易于规模，但清洗可能是困难和可能存在于过滤器的能力造成负面影响。设计的-实验的方法可以被用来识别影响HCP去除和产物回收最相关的工艺参数。这有利于在其他平台上的表达每一种方法和最合适的方法鉴定为给定的纯化策略的应用。

由热烫烟草宿主细胞蛋白质热沉淀 在第2节中描述的漂白过程已成功用于从烟草与70℃的叶，96±1％减少对TSP沉淀医疗专业人员（N = 3），而回收到的Vax8靶蛋白的51％，从而增加了它的为0.1％至1.2％的纯度的色谱分离16之前。它也可以恢复荧光蛋白DsRed的83±1％（N = 3），从3.3％增加其纯度64.1％。热烫步骤容易地集成到一个标准的提取和澄清方案包括生物?...

Watch this Scientific Journal Video about Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts at JoVE.com

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

之前的任何其他纯化步骤三个换热沉淀方法都能够有效地除去90％以上的烟草宿主细胞蛋白质（医疗专业人员）的提取物。植物医疗专业人员不可逆聚合在温度高于60℃。

通过热烫完整植株或提取物热处理烟草宿主细胞蛋白去除方法的比较

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...

comparison-tobacco-host-cell-protein-removal-methods-blanching-intact

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.0K 次观看.  Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V.. 之前的任何其他纯化步骤三个换热沉淀方法都能够有效地除去90％以上的烟草宿主细胞蛋白质（医疗专业人员）的提取物。植物医疗专业人员不可逆聚合在温度高于60℃。 

视频: Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

机器人平台的高通量原生质体分离和转型

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

科研

遗传学

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to possess medicinally relevant biological activity. However, thus far this potential has not translated into a plethora of triterpene-derived drugs in the clinic. This is arguably (at least partially) a consequence of limited practical synthetic access to this class of compound, a problem that can stifle the exploration of structure-activity relationships and development of lead candidates by traditional medicinal chemistry workflows. Despite their immense diversity, triterpenes are all derived from a single linear precursor, 2,3-oxidosqualene. Transient heterologous expression of biosynthetic enzymes in N. benthamiana can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products that are not naturally produced by this host. Agro-infiltration is an efficient and simple means of achieving transient expression in N. benthamiana. The process involves infiltration of plant leaves with a suspension of Agrobacterium tumefaciens carrying the expression construct(s) of interest. Co-infiltration of an additional A. tumefaciens strain carrying an expression construct encoding an enzyme that boosts precursor supply significantly increases yields. After a period of five days, the infiltrated leaf material can be harvested and processed to extract and isolate the resulting triterpene product(s). This is a process that is linearly and reliably scalable, simply by increasing the number of plants used in the experiment. Herein is described a protocol for rapid preparative-scale production of triterpenes utilizing this plant-based platform. The protocol utilizes an easily replicable vacuum infiltration apparatus, which allows the simultaneous infiltration of up to four plants, enabling batch-wise infiltration of hundreds of plants in a short period of time.

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

Transient heterologous expression of biosynthetic enzymes in Nicotiana benthamiana leaves can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products. Herein is described a detailed protocol for rapid (5 days) preparative-scale production of triterpenes and analogs utilizing this powerful plant-based platform.

烟草 Benthamiana叶在制备鳞片上的瞬态表达

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to ...

生物化学

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

烟草中瞬时表达的蛋白作为一个案例研究：复杂系统采用的实验方法设计的表征

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

生物工程

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall process costs. Alternative, cost-effective capture steps are therefore valuable for industrial-scale manufacturing, where large quantities of a single mAb are produced. Here we present a method for the immobilization of a DsRed-based epitope ligand to a cross-linked agarose resin allowing the selective capture of the HIV-neutralizing antibody 2F5 from crude plant extracts without using Protein A. The linear epitope ELDKWA was first genetically fused to the fluorescent protein DsRed and the fusion protein was expressed in transgenic tobacco (Nicotiana tabacum) plants before purification by immobilized metal-ion affinity chromatography. Furthermore, a method based on activated cross-linked agarose was optimized for high ligand density, efficient coupling and low costs. The pH and buffer composition and the soluble ligand concentration were the most important parameters during the coupling procedure, which was improved using a design-of-experiments approach. The resulting affinity resin was tested for its ability to selectively bind the target mAb in a crude plant extract and the elution buffer was optimized for high mAb recovery, product activity and affinity resin stability. The method can easily be adapted to other antibodies with linear epitopes. The new resins allow gentler elution conditions than Protein A and could also reduce the costs of an initial capture step for mAb production.

In this procedure, a DsRed-based epitope ligand is immobilized to produce a highly selective affinity resin for the capture of monoclonal antibodies from crude plant extracts or cell culture supernatants, as an alternative to Protein A.

活联甘蔗，用于亲和色谱树脂的快速发展 - 抗体捕获作为案例研究

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...

Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations

Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to increased susceptibility of smokers to microbial infections and tumor incidence. Ex vivo suppression of receptor-mediated immune responses in human peripheral blood mononuclear cells (PBMCs) treated with smoke constituents is an attractive approach to study mechanisms and evaluate the likely long-term effects of exposure to tobacco products. Here, we optimized methods to perform ex vivo assays using PBMCs stimulated by bacterial lipopolysaccharide, a Toll-like receptor-4 ligand. The effects of whole smoke-conditioned medium (WS-CM), a combustible tobacco product preparation (TPP), and nicotine were investigated on cytokine secretion and target cell killing by PBMCs in the ex vivo assays. We show that secreted cytokines IFN-&#947;, TNF, IL-10, IL-6, and IL-8 and intracellular cytokines IFN-&#947;, TNF-&#945;, and MIP-1&#945; were suppressed in WS-CM-exposed PBMCs. The cytolytic function of effector PBMCs, as determined by a K562 target cell killing assay was also reduced by exposure to WS-CM; nicotine was minimally effective in these assays. In summary, we present a set of improved assays to evaluate the effects of TPPs in ex vivo assays, and these methods could be readily adapted for testing other products of interest.

Using optimized human peripheral blood mononuclear cell (PBMC) ex vivo assays, we showed that a combustible tobacco product preparation markedly suppresses receptor-mediated intracellularly secreted cytokines and cytolytic ability of effector PBMCs. These rapid assays may be useful in product evaluation and understanding the potential long-term effects of tobacco exposure.

方法评估细胞毒性的可燃烟草产品的准备和免疫抑制

Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to ...

免疫与感染

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

优化和利用<em&gt;农杆菌</em&gt;介导的瞬态蛋白生产中<em&gt;烟草</em

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

生物学

Assaying Proteasomal Degradation in a Cell-free System in Plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.
The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.

Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular &#8220;tags&#8221; for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

测定蛋白酶体降解的植物无细胞系统

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

蛋白质 - 蛋白质相互作用的双分子荧光互补的烟草原生质体和叶片可视化

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the flexible, low-cost production of high-quality, bioactive products.
In this study, we compared the expression of a target human recombinant protein in traditional fermenter-based cell cultures (bacterial and insect) with plant-based expression systems, both transient and stable.
For each platform, we described the set-up, optimization and length of the production process, the final product quality and the yields and we evaluated provisional production costs, specific for the selected target recombinant protein.
Overall, our results indicate that bacteria are unsuitable for the production of the target protein due to its accumulation within insoluble inclusion bodies. On the other hand, plant-based systems are versatile platforms that allow the production of the selected protein at lower-costs than Baculovirus/insect cell system. In particular, stable transgenic lines displayed the highest-yield of the final product and transient expressing plants the fastest process development. However, not all recombinant proteins may benefit from plant-based systems but the best production platform should be determined empirically with a case-by-case approach, as described here.

In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.

细菌，昆虫细胞和植物系统:重组蛋白表达在不同的生物工厂的比较分析

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the ...

Screening of Tobacco Genotypes for Phytophthora nicotianae Resistance

Black shank, caused by the oomycetes Phytophthora nicotianae, is destructive to tobacco, and this pathogen is highly pathogenic to many solanaceous crops. P. nicotianae is well adapted to high temperatures; therefore, research on this pathogen is gaining importance in agriculture worldwide because of global warming. P. nicotianae-resistant varieties of tobacco plants are commonly screened by inoculation with oat grains colonized by P. nicotianae and monitoring for the disease symptoms. However, it is difficult to quantify the inoculation intensity since accurate inoculation is crucial in this case. This study aimed to develop an efficient and reliable method for evaluating the resistance of tobacco to infection with P. nicotianae. This method has been successfully used to identify resistant varieties, and the inoculation efficiency was confirmed by real-time PCR. The resistance evaluation method presented in this study is efficient and practical for precision breeding, as well as molecular mechanism research.

Screening of Tobacco Genotypes for Phytophthora nicotianae Resistance

Here, a protocol is presented for the efficient and accurate screening of tobacco genotypes for Phytophthora nicotianae resistance in seedlings. This is a practical approach for precision breeding, as well as molecular mechanism research.

烟草基因型对 烟碱性植物 的筛选

Black shank, caused by the oomycetes Phytophthora nicotianae, is destructive to tobacco, and this pathogen is highly pathogenic to many solanaceous crops. ...

通过热烫完整植株或提取物热处理烟草宿主细胞蛋白去除方法的比较

摘要

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此视频中的章节

机器人平台的高通量原生质体分离和转型

烟草 Benthamiana叶在制备鳞片上的瞬态表达

烟草中瞬时表达的蛋白作为一个案例研究：复杂系统采用的实验方法设计的表征

活联甘蔗，用于亲和色谱树脂的快速发展 - 抗体捕获作为案例研究

方法评估细胞毒性的可燃烟草产品的准备和免疫抑制

优化和利用

测定蛋白酶体降解的植物无细胞系统

蛋白质 - 蛋白质相互作用的双分子荧光互补的烟草原生质体和叶片可视化

细菌，昆虫细胞和植物系统:重组蛋白表达在不同的生物工厂的比较分析

烟草基因型对烟碱性植物的筛选

通过热烫完整植株或提取物热处理烟草宿主细胞蛋白去除方法的比较

摘要

探索更多视频

此视频中的章节

机器人平台的高通量原生质体分离和转型

烟草 Benthamiana叶在制备鳞片上的瞬态表达

烟草中瞬时表达的蛋白作为一个案例研究：复杂系统采用的实验方法设计的表征

活联甘蔗，用于亲和色谱树脂的快速发展 - 抗体捕获作为案例研究

方法评估细胞毒性的可燃烟草产品的准备和免疫抑制

优化和利用

测定蛋白酶体降解的植物无细胞系统

蛋白质 - 蛋白质相互作用的双分子荧光互补的烟草原生质体和叶片可视化

细菌，昆虫细胞和植物系统:重组蛋白表达在不同的生物工厂的比较分析

烟草基因型对 烟碱性植物 的筛选

烟草基因型对烟碱性植物的筛选