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

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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...

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...

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

Research

JoVE Journal

Bioengineering

16.4K 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 ...

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

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