作者感谢托马斯博士拉特马赫提供的PPAM植物表达载体和易卜拉欣Amedi培养在这项研究中所使用的烟草植株。我们要感谢理查德·泰曼博士的协助编辑稿件。这项工作是部分资金由欧洲研究理事会高级格兰特“未来医药”，提案编号269110和Fraunhofer Zukunftsstiftung（弗劳恩霍夫未来基金会）。

<ol><li>Fischer, R., Emans, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+farming+of+pharmaceutical+proteins%5BTitle%5D)+AND+%22Transgenic+research%22%5BJournal%5D)">Molecular farming of pharmaceutical proteins.</a> Transgenic research. 9, 277-299 (2000).</li>
<li>Commandeur, U., Twyman, R. M., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+biosafety+of+molecular+farming+in+plants%5BTitle%5D)+AND+%22AgBiotechNet%22%5BJournal%5D)">The biosafety of molecular farming in plants.</a> AgBiotechNet. 5, 9 (2003).</li>
<li>Shoji, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+plant-based+system+for+rapid+production+of+influenza+vaccine+antigens%5BTitle%5D)+AND+%22Influenza+Other+Resp%22%5BJournal%5D)">A plant-based system for rapid production of influenza vaccine antigens.</a> Influenza Other Resp. 6, 204-210 (2012).</li>
<li>Goodin, M. M., Zaitlin, D., Naidu, R. A., Lommel, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nicotiana+benthamiana%3A+Its+history+and+future+as+a+model+for+plant-pathogen+interactions%5BTitle%5D)+AND+%22Mol+Plant+Microbe+In%22%5BJournal%5D)">Nicotiana benthamiana: Its history and future as a model for plant-pathogen interactions.</a> Mol Plant Microbe In. 21, 1015-1026 (2008).</li>
<li>Berg, R. H., Beachy, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescent+protein+applications+in+plants%5BTitle%5D)+AND+%22Method+Cell+Biol%22%5BJournal%5D)">Fluorescent protein applications in plants.</a> Method Cell Biol. 85, 153 (2008).</li>
<li>Chung, S. M., Vaidya, M., Tzfira, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Agrobacterium+is+not+alone%3A+gene+transfer+to+plants+by+viruses+and+other+bacteria%5BTitle%5D)+AND+%22Trends+in+plant+science%22%5BJournal%5D)">Agrobacterium is not alone: gene transfer to plants by viruses and other bacteria.</a> Trends in plant science. 11, 1-4 (2006).</li>
<li>Sheludko, Y. V., Sindarovska, Y. R., Gerasymenko, I. M., Bannikova, M. A., Kuchuk, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+several+Nicotiana+species+as+hosts+for+high-scale+Agrobacterium-mediated+transient+expression%5BTitle%5D)+AND+%22Biotechnology+and+Bioengineering%22%5BJournal%5D)">Comparison of several Nicotiana species as hosts for high-scale Agrobacterium-mediated transient expression.</a> Biotechnology and Bioengineering. 96, 608-614 (2007).</li>
<li>Wydro, M., Kozubek, E., Lehmann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+transient+Agrobacterium-mediated+gene+expression+system+in+leaves+of+Nicotiana+benthamiana%5BTitle%5D)+AND+%22Acta+Biochimica+Polonica%22%5BJournal%5D)">Optimization of transient Agrobacterium-mediated gene expression system in leaves of Nicotiana benthamiana.</a> Acta Biochimica Polonica. 53, 289-298 (2006).</li>
<li>Buyel, J. F., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Processing+heterogeneous+biomass%3A+Overcoming+the+hurdles+in+model+building%5BTitle%5D)+AND+%22Bioengineered%22%5BJournal%5D)">Processing heterogeneous biomass: Overcoming the hurdles in model building.</a> Bioengineered. 4, (2013).</li>
<li>Fischer, R., Schillberg, S., Hellwig, S., Twyman, R. M., Drossard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GMP+issues+for+recombinant+plant-derived+pharmaceutical+proteins%5BTitle%5D)+AND+%22Biotechnol+Adv%22%5BJournal%5D)">GMP issues for recombinant plant-derived pharmaceutical proteins.</a> Biotechnol Adv. 30, 434-439 (2012).</li>
<li>Daniel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((One-at-a-time+plans%5BTitle%5D)+AND+%22Journal+of+the+American+Statistical+Association%22%5BJournal%5D)">One-at-a-time plans.</a> Journal of the American Statistical Association. 68, 353-360 (1973).</li>
<li>Czitrom, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((One-Factor-at-a-Time+versus+Designed+Experiments+The+American+Statistician%5BTitle%5D)+AND+%22%22%5BJournal%5D)">One-Factor-at-a-Time versus Designed Experiments The American Statistician.</a> 53, 6 (1999).</li>
<li>Anderson, M. J., Kraber, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Keys+to+successful+designed+experiments%5BTitle%5D)+AND+%22ASQ+-+The+global+voice+of+quality%22%5BJournal%5D)">Keys to successful designed experiments.</a> ASQ - The global voice of quality. 6, 6 (1999).</li>
<li>Montgomery, D. C. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMontgomery%2C+DC+%22Design+and+Analysis+of+Experiments%22">Design and Analysis of Experiments</a>. , John Wiley & Sons Inc. (2007).</li>
<li>Myers, R. H., Montgomery, D. C., Anderson-Cook, C. M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMyers%2C+RH+%22Response+Surface+Methodology%3A+Process+and+Product+Optimization+Using+Designed+Experiments%22">Response Surface Methodology: Process and Product Optimization Using Designed Experiments</a>. , Wiley. (2009).</li>
<li>Piepel, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Programs+for+generating+extreme+vertices+and+centroids+of+linearly+constrained+experimental+regions%5BTitle%5D)+AND+%22J+Qual+Technol%22%5BJournal%5D)">Programs for generating extreme vertices and centroids of linearly constrained experimental regions.</a> J Qual Technol. 20, 15 (1988).</li>
<li>FDA. , U.S. Department of Health and Human Services. Rockville, MD, USA. (2009).</li>
<li>Shivhare, M., McCreath, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Practical+Considerations+for+DoE+Implementation+in+Quality+By+Design%5BTitle%5D)+AND+%22BioProcess+International%22%5BJournal%5D)">Practical Considerations for DoE Implementation in Quality By Design.</a> BioProcess International. 8, 9 (2010).</li>
<li>Buyel, J. F., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Predictive+models+for+transient+protein+expression+in+tobacco+%28Nicotiana+tabacum+L.%29+can+optimize+process+time%2C+yield%2C+and+downstream+costs%5BTitle%5D)+AND+%22Biotechnology+and+bioengineering%22%5BJournal%5D)">Predictive models for transient protein expression in tobacco (Nicotiana tabacum L.) can optimize process time, yield, and downstream costs.</a> Biotechnology and bioengineering. 109, 2575-2588 (2012).</li>
<li>Buyel, J. F., Kaever, T., Buyel, J. J., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Predictive+models+for+the+accumulation+of+a+fluorescent+marker+protein+in+tobacco+leaves+according+to+the+promoter%2F5%27UTR+combination%5BTitle%5D)+AND+%22Biotechnology+and+bioengineering%22%5BJournal%5D)">Predictive models for the accumulation of a fluorescent marker protein in tobacco leaves according to the promoter/5'UTR combination.</a> Biotechnology and bioengineering. 110, 471-482 (2013).</li>
<li>Anderson, M. J., Whitcomb, P. J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aAnderson%2C+MJ+%22DOE+Simplified%3A+Practical+Tools+for+Effective+Experimentation%22">DOE Simplified: Practical Tools for Effective Experimentation</a>. , Productivity Inc. (2000).</li>
<li>Anderson, M. J., Whitcomb, P. J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aAnderson%2C+MJ+%22Response+Surface+Methods+Simplified%22">Response Surface Methods Simplified</a>. , Productivity Press. (2005).</li>
<li>De Gryze, S., Langhans, I., Vandebroek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+the+correct+intervals+for+prediction%3A+A+tutorial+on+tolerance+intervals+for+ordinary+least-squares+regression%5BTitle%5D)+AND+%22Chemometr+Intell+Lab%22%5BJournal%5D)">Using the correct intervals for prediction: A tutorial on tolerance intervals for ordinary least-squares regression.</a> Chemometr Intell Lab. 87, 147-154 (2007).</li>
<li> Plasmid DNA purification User manual. , MACHEREY-NAGEL. Düren. (2012).</li>
<li> PCR clean-up Gel extraction User manual. , MACHEREY-NAGEL. Düren. (2012).</li>
<li>Quick Ligation Protocol. 4, NewEnglandBiolabs. Ipswich. (2009).</li>
<li>Inoue, H., Nojima, H., Okayama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-Efficiency+Transformation+of+Escherichia-Coli+with+Plasmids%5BTitle%5D)+AND+%22Gene%22%5BJournal%5D)">High-Efficiency Transformation of Escherichia-Coli with Plasmids.</a> Gene. 96, 23-28 (1990).</li>
<li>Main, G. D., Reynolds, S., Gartland, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electroporation+protocols+for+Agrobacterium%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Electroporation protocols for Agrobacterium.</a> Methods in Molecular Biology. 44, 405-412 (1995).</li>
<li>Gruber, A. R., Lorenz, R., Bernhart, S. H., Neubock, R., Hofacker, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Vienna+RNA+websuite%5BTitle%5D)+AND+%22Nucleic+acids+research%22%5BJournal%5D)">The Vienna RNA websuite.</a> Nucleic acids research. 36, 70-74 (2008).</li>
<li>Howell, S., Kenmore, M., Kirkland, M., Badley, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-density+immobilization+of+an+antibody+fragment+to+a+carboxymethylated+dextran-linked+biosensor+surface%5BTitle%5D)+AND+%22J+Mol+Recognit%22%5BJournal%5D)">High-density immobilization of an antibody fragment to a carboxymethylated dextran-linked biosensor surface.</a> J Mol Recognit. 11, 200-203 (1998).</li>
<li>Newcombe, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+a+biosensor+assay+to+quantify+polyclonal+IgG+in+ovine+serum+used+for+the+production+of+biotherapeutic+antibody+fragments%5BTitle%5D)+AND+%22Process+Biochem%22%5BJournal%5D)">Evaluation of a biosensor assay to quantify polyclonal IgG in ovine serum used for the production of biotherapeutic antibody fragments.</a> Process Biochem. 41, 842-847 (2006).</li>
<li>Peixoto, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hierarchical+Variable+Selection+in+Polynomial+Regression-Models%5BTitle%5D)+AND+%22Am+Stat%22%5BJournal%5D)">Hierarchical Variable Selection in Polynomial Regression-Models.</a> Am Stat. 41, 311-313 (1987).</li>
<li>Peixoto, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Property+of+Well-Formulated+Polynomial+Regression-Models%5BTitle%5D)+AND+%22Am+Stat%22%5BJournal%5D)">A Property of Well-Formulated Polynomial Regression-Models.</a> Am Stat. 44, 26-30 (1990).</li>
<li>Sanders, P. R., Winter, J. A., Barnason, A. R., Rogers, S. G., Fraley, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+cauliflower+mosaic+virus+35S+and+nopaline+synthase+promoters+in+transgenic+plants%5BTitle%5D)+AND+%22Nucleic+acids+research%22%5BJournal%5D)">Comparison of cauliflower mosaic virus 35S and nopaline synthase promoters in transgenic plants.</a> Nucleic acids research. 15, 1543-1558 (1987).</li>
<li>Ma, J. K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+and+Assembly+of+Secretory+Antibodies+in+Plants%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Generation and Assembly of Secretory Antibodies in Plants.</a> Science. 268, 716-719 (1995).</li>
<li>Wycoff, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Secretory+IgA+antibodies+from+plants%5BTitle%5D)+AND+%22Curr+Pharm+Design%22%5BJournal%5D)">Secretory IgA antibodies from plants.</a> Curr Pharm Design. 11, 2429-2437 (2005).</li>
<li>Pace, C. N., Vajdos, F., Fee, L., Grimsley, G., Gray, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+to+measure+and+predict+the+molar+absorption+coefficient+of+a+protein%5BTitle%5D)+AND+%22Protein+Sci%22%5BJournal%5D)">How to measure and predict the molar absorption coefficient of a protein.</a> Protein Sci. 4, 2411-2423 (1995).</li>
</ol>

该刊物的费用部分由公司Statease公司（美国）及STATCON（德国），这些都没有涉及参与的手稿或承担任何其内容的准备赞助。

每个实验需要仔细的规划，因为资源通常是稀缺和昂贵。这是能源部战略尤其如此，因为在规划阶段（ 如选择基本模型，它并不包括所有显著因素相互作用）的错误可以大大减少所产生的模型的预测能力，从而贬低了整个实验。然而，可以很容易地通过以下方式避免基本程序这些错误。 在能源部的规划注意事项首先，要选择一个响应，适合于评估结果（正或负）的每个能源部运行是很重要的。例如：通过紫外吸收37所确定的目标蛋白的浓度是非常有用的，以评估一个特定的启动子/ 5&#39;UTR组合的活性。然而，蛋白质的功能性评估（ 例如或由荧光红色荧光蛋白的情况下，结合在中科院蛋白A2G12电子），甚至更好，因为它也包括一个蛋白质的质量方面。反应也可以从两个或多个单独的参数，例如电源数NP在发酵实验（从电源输入计算出的，旋转速度和搅拌器的直径）计算的值。响应应该产生价值，精度高（ 即低标准差）和准确性（ 即零个或几个干扰参数），以提高模型的质量。合适的检测装置和方法，因此应如有必要​​事先优化实验。 影响反应的因素必须被识别之前，能源部被实现，因为RSM用于确定各因素的确切影响，而不是将其选中。与上响应显著影响因素的选择可以通过研究文献资料来实现，但全部或部分因子能源部策略更有效。这些筛选实验应也可以用来确定一个范围的每个要素中的有意义的结果可以实现， 例如 ，在生物系统中的因子“温度”通常限定在范围10-40℃。离散电平然后应这些范围内定义在技术上难以控制数值的因素， 如人工气候室的温度为±2.0℃的误差，因此是不合适的调查±1.0℃，温差实际的限制也可能开始发挥作用， 例如使用&gt; 25不同浓度的特定化合物。但是，级别的数量必须比因素的基本模型， 如预期的多项式阶数至少有一个更大，如果温度的多项式阶数为n = 2，则至少有三个温度水平，必须予以追究。采用高多项式阶数（立方或以上）的基本型号是一种选择，如果一个因素的多项式顺序是不确定的。然而，这可以显著提高所需的DoE试验次数。 对于复杂系统与许多因素和高多项式订单这可能导致不能有效地计算设计。在这种情况下，它可能是可取的分裂问题分成两个或更多的设计与调查的因素的不同子集。然而，显著因素，从这种“分割设计”省略必须仔细调整为固定值，以防止任何结果的失真。每个分割的设计应反复在不同设定为省略的因素，揭示它们与包括在设计的因素之间的相互作用。这是有益的，以确定该因素对反应的影响最大，并将其包含在所有的设计。 在能源部评估的关键环节该FDS应该在一个新能源部计算评估的第一个参数。操作员必须确认日Ë能源部提供的设计空间所需的最低检测差异和响应的估计标准差的必要保障。如果不是这种情况下，设计时应增大与运行并重复适当数目，直到满足要求。计算能源部（DoE）运行的程序可以被初始化多次与最优性的损失最小设计选择，如果启用了明确的因素之间的平衡。 数据转换应该被执行，如果响应数值差距超过一个数量级。 A登入10改造往往是适当的，但包装盒-考克斯情节会建议，将产生最稳定的模式转型。在这个图所建议的转化可能会改变，一旦最后的模式已经被选择，因此应当重新计算。仅显著因子（p值&lt;0.05），应包括在响应面模型，以保证它的预测p奥尔。例外，可向维护模型层次32,33或以包括已知有基于先前的结果产生影响的因素。在后一种情况下，最好是研究为什么没有找到这样的重要因素是在当前的DoE显著。不适配的参数可以与R平方值组合使用（ 表2和表3）的快速检测不良模型/千篇一律的，而调整后的R平方值指示是否太多/微不足道的因素已选定和预测的R平方是该模型的预测能力的一个指标。然而，在DesignExpert的诊断部分提供的残差图是更重要的，以评估模型的质量，因为它们允许趋势数据集显示的隐藏因素的存在，以及极端值的鉴别检测。后者对合适的高于平均水平的冲击和扭曲模型。这样的值可以来自有缺陷的数据（ 如置换值），必须予以纠正，或从失败的实验，必须重复。如果一个极端值被发现是“真正的”在这样的反复实验中，建议增加几个运行在这个值的接近，提高了设计空间的这个区域内的模型的质量。 修正设计方案，这是不完善的或太小不管为什么加班可能需要（ 如丢失数据，无法运行，真正的极值和地区，未预料到的高阶因素相互作用），这些试验应该使用“设计增强”工具被添加到一个有序的方式对现有设计介绍在一个单独的块中的新线路。这样做将有利于使用以前的数据集，同时选择最有意义的加班。其结果将是一个适合于该数据，并允许可靠的模型预测未来的结果。

生物的蛋白质在植物中的产量是有益的，因为植物是便宜的增长，该平台可以扩大刚刚通过生长得更植物和人类病原体无法复制1,2。例如基于与叶根癌农杆菌浸润瞬时表达策略提供了额外的好处，因为DNA传递和净化产品的交付点之间的时间减少从几年到不到2个月3。瞬时表达也可用于功能分析， 例如来测试基因的互补功能丧失的突变体或以调查蛋白质相互作用4-6能力。然而，瞬时表达水平往往表现出更大的批与批之间的变化比在转基因植物中7-9的表达水平。这减少了基于瞬时表达的Wi生物制药生产过程的可能性会批准在良好生产规范（GMP）的情况下，因为再现性是一个关键质量属性，并受风险评估10。这种变化也可以掩盖，研究人员打算调查的任何相互作用。因此，我们开始着手找出影响植物中瞬时表达水平，打造一支高素质的定量预测模型的主要因素。 一个因子在每次一个（OFAT）方法经常用于表征某些参数（ 系数 ）的对结果的实验11的影响（ 效应 ）（ 响应 ）。但是，这是不理想的，因为单独的测试（ 运行 ）的调查（ 实验 ）期间将通过被测试（ 设计空间 ）的因素跨越的潜力区对齐像一串珍珠。设计空间和信息，因此从实验中得出的程度的覆盖范围是低， 如图1A 12。此外，不同的因素（ 因子相互作用 ）之间的相互依赖关系可以保持隐蔽导致差的模型和/或假最优的预测， 如图1B 13。 上述缺点，可避免使用试验设计（DOE）的方法，其中一个实验的运行被整个设计空间散布更加均匀地设计，这意味着多于一个因素是两个运行14之间变化。有专门设计的混合物，筛选因子（ 因子设计 ）和对反应的影响因素的定量（ 响应面方法，RSM次）15。此外，的RSM可被实现为中央复合设计，但也可以通过使用能够对运行中的选择应用不同的标准专门的软件有效地实现。例如，所谓的D-optimalitŸ标准将选择运行，以便在生成的模型的系数，以尽量减少误差，而IV-最优标准选择运行的实现整个设计空间15,16最低的预测方差。我们在这里描述的RSM允许在植物中瞬时表达的精确定量，但是它可以很容易地转移到涉及多个（〜5-8）的任何系统的数值的因素（ 如温度，时间，浓度）和几个（〜2 - 4） 明确的因素（ 如启动子，颜色），其中一种机械的描述不可用或过于复杂的模型。 能源部的方法起源于农业科学，但已经扩散到其它区域，因为它是可转移到任何情况下它减少必要获得可靠的数据运行的次数，并生成描述性模型为复杂的过程是有用的。这反过来又导致了“指引纳入能源部的业，Q8（R2）医药发展“通过对技术要求国际协调会议公布了注册人用药品（ICH）17。能源部目前广泛应用于科研和工业18。然而，护理过程中必须采取规划和执行实验，因为选择不当的多项式程度的多重线性回归模型（ 基本型号 ）可以引入一个需要额外的运行，以正确模拟所有因素的影响。此外，损坏或丢失的数据产生不正确的型号和有缺陷预测，甚至可能防止在协议和讨论第18条所述的任何模型建立的尝试。在协议中，我们将首先列出最重要的规划步骤为RSM为基础的实验，然后解释根据能源部的设计软件DesignExpert V8.1，但类似的设计可以与其他软件includi建吴JMP，Modde和STATISTICA。实验步骤之后是对数据进行分析和评价的指示。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51216/51216fig1highres.jpg" src="/files/ftp_upload/51216/51216fig1.jpg" /> 图1。 OFAT和能源部，一个比较 。的一个因素在一个实验（黑，红和蓝色圆圈）连续变化的时间（OFAT）实现了设计空间（斜线区域）的低覆盖率。与此相反，一个以上的因素在一个时间使用的试验设计（DOE）的策略（绿色圆圈）的设计的变化提高了覆盖率，将所得模型。 乙因而精度。偏置设计空间覆盖意味着OFAT实验（黑圈）也可能失败，找出最佳的操作区域（红色）和预测次优的解决方案（大黑圆），而能源部将军会ES（黑星）更容易找出最好的条件（大黑星）。

<table><tbody><tr><td>Design-Expert(R) 8</td><td>Stat-Ease, Inc.</td><td>n.a.</td><td>DoE software</td></tr><tr><td>Tryptone</td><td>Carl Roth GmbH</td><td>8952.2</td><td>Media component</td></tr><tr><td>Yeast extract</td><td>Carl Roth GmbH</td><td>2363.2</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>Ampicillin</td><td>Carl Roth GmbH</td><td>K029.2</td><td>Antibiotic</td></tr><tr><td>Agar-Agar</td><td>Carl Roth GmbH</td><td>5210.2</td><td>Media component</td></tr><tr><td>&lt;i&gt;Escherichia coli&lt;/i&gt; K12 DH5a</td><td>Life Technologies</td><td>18263-012</td><td>Microorganism</td></tr><tr><td>pPAM</td><td>GenBank</td><td>AY027531</td><td>Cloning/expression vector;&amp;nbsp;</td></tr><tr><td>NucleoSpin Plasmid&amp;nbsp;</td><td>MACHEREY-NAGEL GmbH</td><td>740588.250</td><td>Plasmid DNA isolation kit</td></tr><tr><td>NucleoSpin Gel and PCR Clean-up</td><td>MACHEREY-NAGEL GmbH</td><td>740609.250</td><td>Plasmid DNA purification kit</td></tr><tr><td>NanoDrop 2000</td><td>Thermo Scientific</td><td>n.a.</td><td>Spectrophotometer</td></tr><tr><td>NcoI</td><td>New England Biolabs Inc.</td><td>R3193L</td><td>Restrictionendonuclease</td></tr><tr><td>EcoRI</td><td>New England Biolabs Inc.</td><td>R3101L</td><td>Restrictionendonuclease</td></tr><tr><td>AscI</td><td>New England Biolabs Inc.</td><td>R0558L</td><td>Restrictionendonuclease</td></tr><tr><td>NEB 4</td><td>New England Biolabs Inc.</td><td>B7004S</td><td>Restrictionendonuclease buffer</td></tr><tr><td>TRIS</td><td>Carl Roth GmbH</td><td>4855.3</td><td>Media component</td></tr><tr><td>Disodium tetraborate</td><td>Carl Roth GmbH</td><td>4403.3</td><td>Media component</td></tr><tr><td>EDTA</td><td>Carl Roth GmbH</td><td>8040.2</td><td>Media component</td></tr><tr><td>Agarose</td><td>Carl Roth GmbH</td><td>6352.4</td><td>Media component</td></tr><tr><td>Bromophenol blue</td><td>Carl Roth GmbH</td><td>A512.1</td><td>Color indicator</td></tr><tr><td>Xylene cyanol</td><td>Carl Roth GmbH</td><td>A513.1</td><td>Color indicator</td></tr><tr><td>Glycerol</td><td>Carl Roth GmbH</td><td>7530.2</td><td>Media component</td></tr><tr><td>Mini-Sub Cell GT Cell</td><td>BioRad</td><td>170-4406</td><td>Gel electrophoresis chamber</td></tr><tr><td>&lt;i&gt;Agrobacterium tumefaciens&lt;/i&gt; strain GV3101:pMP90RK</td><td>DSMZ</td><td>12365</td><td>Microorganism</td></tr><tr><td>Electroporator 2510</td><td>Eppendorf</td><td>4307000.658</td><td>Electroporator</td></tr><tr><td>Beef extract</td><td>Carl Roth GmbH</td><td>X975.2</td><td>Media component</td></tr><tr><td>Peptone</td><td>Carl Roth GmbH</td><td>2365.2</td><td>Media component</td></tr><tr><td>Sucrose</td><td>Carl Roth GmbH</td><td>4621.2</td><td>Media component</td></tr><tr><td>Magnesium sulfate</td><td>Carl Roth GmbH</td><td>0261.3</td><td>Media component</td></tr><tr><td>Carbenicillin</td><td>Carl Roth GmbH</td><td>6344.2</td><td>Antibiotic</td></tr><tr><td>Kanamycin</td><td>Carl Roth GmbH</td><td>T832.3</td><td>Antibiotic</td></tr><tr><td>Rifampicin</td><td>Carl Roth GmbH</td><td>4163.2</td><td>Antibiotic</td></tr><tr><td>FWD primer</td><td>Eurofins MWG Operon</td><td>n.a.</td><td>CCT CAG GAA GAG CAA TAC</td></tr><tr><td>REV primer</td><td>Eurofins MWG Operon</td><td>n.a.</td><td>CCA AAG CGA GTA CAC AAC</td></tr><tr><td>2720 Thermal cycler</td><td>Applied Biosystems</td><td>4359659</td><td>Thermocycler</td></tr><tr><td>RNAfold webserver</td><td>University of Vienna</td><td>n.a.</td><td>Software</td></tr><tr><td>Ferty 2 Mega</td><td>Kammlott</td><td>5.220072</td><td>Fertilizer</td></tr><tr><td>Grodan Rockwool Cubes 10 x10 cm</td><td>Grodan</td><td>n.a.</td><td>Rockwool block</td></tr><tr><td>Greenhouse</td><td>n.a.</td><td>n.a.</td><td>For plant cultivation</td></tr><tr><td>Phytotron</td><td>Ilka Zell</td><td>n.a.</td><td>For plant cultivation</td></tr><tr><td>Omnifix-F Solo</td><td>B. Braun</td><td>6064204</td><td>Syringe</td></tr><tr><td>Murashige and Skoog salts</td><td>Duchefa</td><td>M 0222.0010</td><td>Media component</td></tr><tr><td>Glucose</td><td>Carl Roth GmbH</td><td>6780.2</td><td>Media component</td></tr><tr><td>Acetosyringone</td><td>Sigma-Aldrich</td><td>D134406-5G</td><td>Phytohormon analogon</td></tr><tr><td>&amp;nbsp;BioPhotometer plus</td><td>Eppendorf</td><td>6132 000.008</td><td>Photometer</td></tr><tr><td>Osram cool white 36 W</td><td>Osram</td><td>4930440</td><td>Light source</td></tr><tr><td>Disodium phosphate</td><td>Carl Roth GmbH</td><td>4984.3</td><td>Media component</td></tr><tr><td>Centrifuge 5415D</td><td>Eppendorf</td><td>5424 000.410</td><td>Centrifuge</td></tr><tr><td>Forma -86C ULT freezer</td><td>ThermoFisher</td><td>88400</td><td>Freezer</td></tr><tr><td>Synergy HT</td><td>BioTek</td><td>SIAFRT</td><td>Fluorescence plate reader</td></tr><tr><td>Biacore T200</td><td>GE Healthcare</td><td>n.a.</td><td>SPR device</td></tr><tr><td>Protein A</td><td>Life Technologies</td><td>10-1006</td><td>Antibody binding protein</td></tr><tr><td>HEPES</td><td>Carl Roth GmbH</td><td>9105.3</td><td>Media component</td></tr><tr><td>Tween-20</td><td>Carl Roth GmbH</td><td>9127.3</td><td>Media component</td></tr><tr><td>2G12 antibody</td><td>Polymun</td><td>AB002</td><td>Reference antibody</td></tr></tbody></table>

characterization of complex systems using the design of experiments approach: transient protein expression in tobacco as a case study

工厂提供用于生产生物制药，包括成本低，可扩展性和安全性的多重效益。瞬时表达提供短期开发和生产时间的额外优势，但表达水平可以分批从而在良好生产规范的背景下监管问题引起的显著差异。我们使用的试验设计（DOE）的方式设计确定的主要因素，如表达过程中的表达构建体的调节元件，植物生长和发育的参数，并在培养条件下，在表达的批次之间的变异性的影响。我们测试了植物表达模型的抗HIV单克隆抗体（2G12）和荧光标记蛋白（红色荧光蛋白）。我们讨论的理由来选择模型的某些性质，并确定其潜在的局限性。一般的处理方法可以很容易地转移到其他问题，因为该模型的原理再广泛适用：基于知识的参数选择，降低复杂性通过把最初的问题分成更小的模块，优化实验组合软件引导安装程序和阶梯式设计增强。因此，该方法不仅用于表征植物蛋白的表达也为其他复杂系统缺乏一种机械描述的调查有用的。预测方程描述参数之间的互连性可用于建立机理模型用于其它复杂的系统。

使用不同的启动子和5&#39;UTRs过程中瞬时表达红色荧光蛋白积聚的描述性模型 在叶中提取红色荧光蛋白的荧光被用来表明该重组蛋白的表达水平，并因此被用作在能源部策略的响应。我们认为相关的最小可检测差异为20微克/毫升和系统的估计的标准偏差为8微克/毫升的基础上初步实验。包含在瞬时表达模型的因素的基础上文献资料7,8和我们以前的结果9个被选中。所研究的范围分别根据这些数据还选择（ 表1）。至少在三个层次被选定为所有离散数值的因素，使二次基础模型的计算。 A D-最优选择算法被选定为能源部的选择上运行，以获得最准确的估计回归模型的系数。通过DesignExpert最初建议的设计包括90运行，但FDS不足以达到预测（ 图3A）1％的标准误差。设计的D-最优增强合共210运行解决了这个问题，并导致100％，各由平坦的曲线（ 图3B）所示的设计空间更均匀的预测精度的FDS。 的红色荧光蛋白浓度测定所有210道和数据均记录10转化。模型的因素被选为由立方模型的0.100阿尔法级自动化落后的选择。这导致显著模型（ 表2）和高不适配的微不足道值的复相关系数（ 表2）。所有模型因素的p值&lt;0.05，因此没有进一步的手动操作的模式是必需的。该模型包含三因素相互作用所没有的初始二次示范基地的一部分（ 表2）。重新评估使用包含在最终的预测模型中的所有因素FDS图形显示，该FDS进行预测的标准误差并没有显著者包括另外三个因素的相互作用（FDS = 0.99）减少。 在DesignExpert模型质量诊断工具，表明数据转换为有用且没有遗漏的因素在模型中，因为残留的正常表现情节的线性特性，并没有具体的模式，观察的残差与预测图（ 图5A和5B ）。也有在整个实验过程中没有趋势来表示一个隐藏的依赖于时间的变化（ 图5C）。相反，该模型的预测是在与所观察到的红色荧光蛋白的荧光（ 图5D）非常吻合。我们一般herefore假设所选择的模式是有用的预测红色荧光蛋白在期间持续8天，后渗透潜伏期由不同的启动子/ 5&#39;UTR组合驱动的非子叶烟叶的瞬时表达。我们还选择了人工线性回归模型，而数据转换来说明错误的因素选择和变换的后果（ 图6）。显然，残差的正常情节偏离了预期的线性关系（ 图6A），并有一个“V”形图案中的残差与预测的情节而不是随机的散射（ 图6B）。此外，残差与运行的情节突出了两个极值（ 图6C），而预测是差了两个，小的，高价值，从优化模型（对角线）（ 图6D）偏离。 在5&#39;UTR组合与CaMV启动35SS启动导致更强的红色荧光蛋白的表达比组合，带nos启动 （ 图5A和5B）如先前报道34和相应的因素被认为是在能源部模型（ 表2）显著。该模型还预测，叶龄是一个显著的因素（ 表2）显示，以嫩叶较高的表达水平（ 图7B和7D），这与我们以前的研究结果19和别人7,8的一致。红色荧光蛋白积聚在叶片级数不是线性或指数，但随后一个S形曲线中的后8天浸润孵育根据该模型。有趣的是，在5&#39;UTRs在相应的红色荧光蛋白的表达上没有固定的顺序。因此，一个5&#39;UTR的“强度”是依赖于所附的启动子。这是不可能的，这启动子和5&#39;UTR之间的相互依存关系会一直使用OFAT实验揭示。该预测模型也表明，某些双启动子/ 5&#39;UTR组合（ 如 。的CaMV 35SS/CHS及NOS / CHS（ 图7A和7B）的CaMV 35SS/omega及NOS / CHS或CaMV启动35SS/CHS和CaMV启动35SS / TL）导致了一个平衡的表达水平，从各叶片和孵育时间&gt; 2天（ 例如 ，对于CaMV启动35SS/omega 和 nos / CHS比为〜8.0）20定义的比率小于30％的不同。这种均衡的表现将是多聚体蛋白如IgA与定义的化学计量35,36的表达是有用的。 <img alt="图5" fo:content-width="5in" fo:src="/files/ftp_upload/51216/51216fig5highres.jpg" src="/files/ftp_upload/51216/51216fig5.jpg" /> 图5中把握模型质量的HICAL评价。国内学生化残差的，正常概率图表示正态分布，因为残差沿着某条线：B。国内学生化残差围绕零值的预测响应（荧光）的所有范围分散指示适当的数据转换。Ç。内部学生化残差实验的整个过程中围绕零值分散，表现出没有隐藏的时间效应。Ð。预测和响应的实际值有较好的一致性，因为所有的点都靠近对角线（理想模型）。 <img alt="图6" fo:content-width="5in" fo:src="/files/ftp_upload/51216/51216fig6highres.jpg" src="/files/ftp_upload/51216/51216fig6.jpg" /> 图6。诊断表明低质量模型，一个 </str翁&gt;。国内学生化残差的正态概率图表示非正态分布，因为残差从一条线：B偏离。国内学生化残差呈现“V”字形分布，表示不恰当的数据转换。Ç。内部学生化残差实验的整个过程中不分散各处的值为零，但表现出朝向正的值的倾向。此外，两个极端值都可以找到。ð。预测和响应的实际值是在贫穷的协议，因为大部分百分点对角线（理想模型）偏离。 <img alt="图7" fo:content-width="5in" fo:src="/files/ftp_upload/51216/51216fig7highres.jpg" src="/files/ftp_upload/51216/51216fig7.jpg" /> 图7。响应烟叶表面的瞬态DsRed的表达式。 </s<html> 。TRONG&gt; 号 / CHS叶2，B。 CaMV启动35SS/CHS叶2，C。 CaMV启动35SS/TL叶6，D。 CaMV启动35SS/CHS叶6。发病期间在S形的方式红色荧光蛋白的浓度增加。表达水平相比，嫩叶（ 例如叶6中C和D）和NOS（A）相比的CaMV 35SS子（B）于老叶较低（ 例如叶2中A和B）。在5&#39;UTR也对DsRed的表达（ 例如，TL（℃）与 CHS（D）），但表达强度依赖于所附的启动子的显著影响。 <table border="0" cellpadding="0" cellspacing="0" width="384"><tbody><tr height="20"><td height="20" style="height:20px;width:64px;">源</td><td style="width:64px;">平方和</td>“&gt; DF </td><td style="width:64px;">均方</td><td style="width:64px;"> F值</td><td style="width:64px;"> p值</td></tr><tr height="20"><td height="20" style="height:20px;">模型</td><td> 174.85 </td><td> 95 </td><td> 1.84 </td><td> 182.12 </td><td> &lt;0.0001 </td></tr><tr height="20"><td height="20" style="height:20px;">叶位置（A） </td><td> 0.16 </td><td> 1 </td><td> 0.16 </td><td> 16.06 </td><td> 0.0001 </td></tr><tr height="20"><td height="20" style="height:20px;">孵育时间（B） </td><td> 112.46 </td><td> 1 </td><td> 112.46 </td><td> 11128.22 </td><td> &lt;0.0001 </td></tr><tr height="20"><td height="20" style="height:20px;">叶龄（C） </td><td> 15.24 </td><td> 7 </td><td> 2.18 </td><td> 215.39 </td><td> &lt;0.0001 </td></tr><tr height="20"><td height="20" style="height:20px;">发起人（D） </td><td> 23.49 </td><td> 1 </td><td> 23.49 </td><td> 2324。29 </td><td> &lt;0.0001 </td></tr><tr height="20"><td height="20" style="height:20px;"> 5&#39;UTR（E） </td><td> 0.93 </td><td> 3 </td><td> 0.31 </td><td> 30.61 </td><td> &lt;0.0001 </td></tr><tr height="20"><td height="20" style="height:20px;">交流</td><td> 0.24 </td><td> 7 </td><td> 0.034 </td><td> 3.38 </td><td> 0.0026 </td></tr><tr height="20"><td height="20" style="height:20px;"> BC </td><td> 1.46 </td><td> 7 </td><td> 0.21 </td><td> 20.64 </td><td> &lt;0.0001 </td></tr><tr height="20"><td height="20" style="height:20px;"> BD </td><td> 2.27 </td><td> 1 </td><td> 2.27 </td><td> 224.51 </td><td> &lt;0.0001 </td></tr><tr height="20"><td height="20" style="height:20px;"> BE </td><td> 0.87 </td><td> 3 </td><td> 0.29 </td><td> 28.74 </td><td> &lt;0.0001 </td></tr><tr height="20"><td height="20" style="height:20px;">光盘</td><td> 0.29 </td><td> 7 </td><td> 0.042 </td><td>4.11 </td><td> 0.0005 </td></tr><tr height="20"><td height="20" style="height:20px;">行政长官</td><td> 0.43 </td><td> 21 </td><td> 0.021 </td><td> 2.04 </td><td> 0.0093 </td></tr><tr height="20"><td height="20" style="height:20px;"> DE </td><td> 0.48 </td><td> 3 </td><td> 0.16 </td><td> 15.73 </td><td> &lt;0.0001 </td></tr><tr height="20"><td height="20" style="height:20px;"> B2 </td><td> 6.34 </td><td> 1 </td><td> 6.34 </td><td> 627.29 </td><td> &lt;0.0001 </td></tr><tr height="20"><td height="20" style="height:20px;"> BCD码 </td><td> 0.95 </td><td> 7 </td><td> 0.14 </td><td> 13.42 </td><td> &lt;0.0001 </td></tr><tr height="20"><td height="20" style="height:20px;"> BCE </td><td> 0.39 </td><td> 21 </td><td> 0.019 </td><td><strong&gt; 1.83 </td><td> 0.0230 </td></tr><tr height="20"><td height="20" style="height:20px;"> BDE </td><td> 0.16 </td><td> 3 </td><td> 0.054 </td><td> 5.37 </td><td> 0.0017 </td></tr><tr height="20"><td height="20" style="height:20px;"> B2D </td><td> 1.49 </td><td> 1 </td><td> 1.49 </td><td> 147.93 </td><td> &lt;0.0001 </td></tr><tr height="20"><td height="20" style="height:20px;">残留</td><td> 1.15 </td><td> 114 </td><td> 0.010 </td><td></td><td></td></tr><tr height="20"><td height="20" style="height:20px;">不适配的</td><td> 1.08 </td><td> 104 </td><td> 0.010 </td><td> 1.45 </td><td> 0.2669 </td></tr><tr height="20"><td height="20" style="height:20px;">纯误差</td><td> 0.072 </td><td> 10 </td><td> 7.153E-003 </td><td></td><td></td></tr><tr height="20"><td height="20" style="height:20px;">总的相关性</td><td> 176.01 </td><td> 209 </td><td></td><td></td><td></td></tr><tr height="20"><td height="20" style="height:20px;">相关系数</td><td>值</td><td></td><td></td><td></td><td></td></tr><tr height="20"><td height="20" style="height:20px;"> R平方</td><td> 0.9935 </td><td></td><td></td><td></td><td></td></tr><tr height="20"><td height="20" style="height:20px;">调整后的R平方值</td><td> 0.9880 </td><td></td><td></td><td></td><td></td></tr><tr height="20"><td height="20" style="height:20px;">预测R平方</td><td> 0.9770 </td><td></td><td></td><td></td><td></td></tr></tbody></table> 表2。包含在预测因子模型瞬态红色荧光蛋白表达的烟叶使用不同的启动子/ 5&#39; 端非编码区的组合。三因素相互作用以粗体显示。 优化培养条件和单克隆抗体的生产在植物中的瞬时表达收获计划 能源部的方法也被用来优化培养温度，细菌OD600nm值对叶子浸润，植物年龄和收获方案中，用于同时生产单克隆HIV-中和抗体2G12和红色荧光蛋白在烟草19。收获计划，确定哪些叶子被用于蛋白质的提取， 例如六个最上面的叶子。因此，我们建立了一个预测模型为每个蛋白（2G12和红色荧光蛋白）的植物在不同年龄段的表达（年轻=早期萌芽阶段=播种收割后40天，老熟=蕾期采收=在47天看后丁）。那么这四款车型进行了评价，并且包含每个因子建立了共识模型发现在个别机型显著。然后，我们证实了共识的模型仍然是所有初始数据集（ 表3）良好的代表性。当时采用的共识模型来确定最佳的孵化温度（〜22℃，2G12和〜25°C为红色荧光蛋白）和细菌OD 600nm的 （〜1.0）两种蛋白质。这些最佳条件，然后用来预测在所有的树叶（1-8）和叶位（1-4）在年轻人和老年人的植物蛋白浓度。的浓度分布进行了整合与生物量数据19，以产生靶蛋白的从不同的叶子和植物的年龄（ 图8A）中得到的绝对量。绝对量的蛋白质，然后用​​相关的下游成本使我们能够进行成本效益分析，每个叶/株龄的处理相关。这表明年轻的植物是有利的瞬时表达，因为蛋白质在较短的生长周期虽有所降低总体生物量较老厂（ 图8B和8C）达到较高的浓度。我们还发现，处理从老厂所有的叶子比丢弃叶1-3和增加每一批植物来代替（ 图8D）的数量较昂贵。因此，能源部（DoE）为基础的模型是合适的，不仅标记实验的最后一步，也为与其他数据相结合，以促进过程分析的更为复杂的问题。一系列相互连接的机型涵盖了整个生产过程中产生的生物制药蛋白质可能是最终的目标。 <img alt="图8" fo:content-width="5in" fo:src="/files/ftp_upload/51216/51216fig8highres.jpg" src="/files/ftp_upload/51216/51216fig8.jpg" /> 图8。INTEGRAT<html>离子导致绝对的蛋白产量和工艺成本的生物量和蛋白质浓度数据。一，叶生物量和目的蛋白浓度不发展导致2G12的嫩叶，B中的偏颇积累的共线方式。红色荧光蛋白和〜相同数量的65％2G12被发现在年轻的植物相比，旧尽管〜低50％，平均生物量。Ç。这反映在幼苗较高的特异性蛋白的表达。ð。增加的特异表达翻译成降低为蛋白质的生产成本，因为需要较少的生物质进行处理需要更少的温室空间，更少的耗材（ 例如 ，过滤器），少下游设备。 <table border="0" cellpadding="0" cellspacing="0" width="576"><colgroup><col span="9" style="width:48pt" width="64" /></colgroup><tbody><tr height="20" style="height:15.0pt"><html>磅;宽：48点“宽度=”64“&gt;植物年龄[DPS] </td><td align="center" class="xl64" colspan="4" style="width:192pt" width="256"> 40 </td><td align="center" class="xl64" colspan="4" style="width:192pt" width="256"> 47 </td></tr><tr height="20" style="height:15.0pt"><td height="20" style="height:15.0pt">靶蛋白[ - ] </td><td align="center" class="xl65" colspan="2">红色荧光蛋白</td><td align="center" class="xl65" colspan="2"> 2G12 </td><td align="center" class="xl65" colspan="2">红色荧光蛋白</td><td align="center" class="xl65" colspan="2"> 2G12 </td></tr><tr height="20" style="height:15.0pt"><td class="xl63" height="20" style="height:15.0pt">型号[ - ] </td><td class="xl63">初始</td><td class="xl63">共识</td><td class="xl63">初始</td><td class="xl63">共识</td><td class="xl63">初始</td><td class="xl63">共识</td><td class="xl63">初始</td><td class="xl63">共识</td></tr>T“&gt; <td height="20" style="height:15.0pt"> R平方[ - ] </td><td> 0.9829 </td><td> 0.9577 </td><td> 0.9321 </td><td> 0.9099 </td><td> 0.9436 </td><td> 0.9403 </td><td> 0.8826 </td><td> 0.8782 </td></tr><tr height="20" style="height:15.0pt"><td height="20" style="height:15.0pt">调整后的R平方[ - ] </td><td> 0.9711 </td><td> 0.9480 </td><td> 0.9059 </td><td> 0.8893 </td><td> 0.9362 </td><td> 0.9350 </td><td> 0.8716 </td><td> 0.8674 </td></tr><tr height="20" style="height:15.0pt"><td class="xl63" height="20" style="height:15.0pt">预测R平方[ - ] </td><td class="xl63"> 0.9510 </td><td class="xl63"> 0.9336 </td><td class="xl63"> 0.8272 </td><td class="xl63"> 0.8587 </td><td class="xl63"> 0.9254 </td><td class="xl63"> 0.9282 </td><td class="xl63"> 0.8554 </td><td class="xl63"> 0.8516 </td></tr></tbody></table> 表3。初始RS的相关性系数比较M车型和最终达成共识红色荧光蛋白和2G12在烟草中的表达模式。

Watch this Scientific Journal Video about Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study at JoVE.com

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

我们描述的实验方法设计，可以被用来确定与模型的转基因的调控元件，植物生长和发育参数和培养条件对植物中的单克隆抗体和报告蛋白的瞬时表达的影响。

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 ...

characterization-complex-systems-using-design-experiments-approach

Nanyang Technological University

Research

JoVE Journal

Bioengineering

16.5K 次观看.  RWTH Aachen University. 我们描述的实验方法设计，可以被用来确定与模型的转基因的调控元件，植物生长和发育参数和培养条件对植物中的单克隆抗体和报告蛋白的瞬时表达的影响。 

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

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 Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

使用吉布森大会和基因枪烟草中瞬时基因表达

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

环境科学

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as&#160;fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems.

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.

重组纤维素酶Cel5A从表达<em&gt;里氏木霉</em&gt;在烟草

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture ...

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

通过诱导瞬时转染可控离子通道表达

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

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

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 ...

生物学

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 ...

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 ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

成像使用烟草瞬时表达系统MAPK信号通路的空间重组

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...