这项工作是支持的BMBF Forschungsinitiative"BioEnergie 2021 - Forschung献给死去Nutzung冯Biomasse"创优"量身定制燃料生物质"，它是通过卓越计画经费由德国联邦和州政府，以促进科学的集群并在德国大学的研究。

1，生长野生型普通烟草植物<ol><li>成长N.烟草 L。 CV。对土壤佩蒂特哈瓦那SR1植物，放置在合适的花盆装满正常的盆栽土壤发芽烟草种子。 2周之后，苗转移至个别盆。孵育恒22°C（黑暗时期）或25℃（光周期）在温室中的植物和70％的相对空气湿度。在八分之一十六小时的光/暗周期提供照明（ 如飞利浦IP65 400瓦灯泡; 180微摩尔·秒-1·米-2，λ= 400〜700 nm）和水，每天。 </li></ol> TrCel5A和TrCel5A-mCherry的蛋白质2。瞬时表达<ol><li>在无菌条件下，挑Agarobacterium癌农杆菌含有两种pTRAkc-ER-TrCel5A或pTRAkc-ER-TrCel5A-的mCherry并转移到含有10ml酵母提取物锥形瓶中单菌落（GV3101 :: pMP90RK GMR，KMR，RIFR）肉汤（YEB，制备成每制造商的规格）介质和卡那霉素（50毫克/毫升），利福平（50毫克/毫升），和羧苄青霉素（100毫克/毫升）以供选择。孵育36-40小时，在26℃和180转。注意:适当的结构来表达设计是本文的范围，但作为指导，可遵循的结构设计麦克莱恩等[13]和芒罗和Pellham 14的方法。 </li><li>取200μl的各培养物接种到20毫升YEB与上面提到的抗生素浓度和以前一样在相同条件下孵育。 </li><li>经过36-40小时后，通过加入2微升acetosyringon（200μM的最终浓度），加入100μl的40％（重量/体积）的葡萄糖溶液和200微升的1M MES缓冲液pH 5.6。preinduce培养孵育过夜。 </li><li>制备100毫升2×缓冲液浸润（100克/升蔗糖，3.6克/升葡萄糖，8.6 g / L的Murashige和Skoog基础盐的用氢氧化钾调节pH值至5.6）。 </li><li>测量1:5稀释的培养物的OD 600，直到它达到的OD 600为5-6。计算出外径600需要30毫升渗透培养基培养体积。加入15毫升2X渗透缓冲，再加入去离子H 2 O至使缓冲高达〜30毫升。 </li><li>就拿植物温室和使用枪头缺口至5 日叶第三的远轴侧从上缓解渗透。 </li><li>用枪头缺口叶子的背面侧，以缓解渗透。 </li><li>填充注射器渗透介质，并把它（不带针）对先前作出的刻痕之一。支持注射器用手指在近轴叶边。仔细按介质进入脉间叶区的组织。注:浸润后，培养在温室中的植物如上文所述的相同的条件下进行。此外，保持untr同等比例eated，非瞬时表达植株（NTEPs）作为对照，在相同的条件下渗入植物下。 </li></ol>在植物叶片TrCel5A-mCherry的表达3。评估<ol><li>经过4-6天，采取植物渗透与甲农杆菌含有pTRAkc-ER-TrCel5A-的mCherry从温室，并将其放置在一个黑暗的房间荧光分析。 </li><li>可视化的荧光表达TrCel5A-的mCherry，光电厂使用的是便携式光源发射绿光在515 nm处有一个红色滤光片（620-750 nm）的叶节。注:在深度在植物叶片蛋白表达的鉴定可以通过薄切片用的叶子在光镜和荧光显微镜一vibratome和考试来实现。对于这种按照以下步骤操作: <ol><li>切约5×5毫米大小的小部分从叶渗透并嵌入在4％琼脂糖溶于50 mM磷酸盐缓冲液（pH 5.7）。 </li><li>切80-90微米大小使用一个vibratome嵌入式叶节。将它们放置在载玻片上，盖上缓冲或盖玻片，用显微镜在10倍放大倍率荧光检测器，使用相同的波长和过滤上述检查它们。 </li></ol></li></ol> 4。TrCel5A提取烟叶<ol><li>从感染A.植物从切烟叶片到1克的重量大约农杆菌含有pTRAkc-ER-TrCel5A并置于预冷用液氮在研钵。加液氮适量的样品。用预冷杵研磨叶粉。 </li><li>加入2毫升含有1mM苯甲磺酰氟的地面叶物质的磷酸盐缓冲盐水（PBS）中并混合，直到大部分的叶物质是在悬浮液中。离心提取液以15,000×g离心20分钟，在4℃，将含有蛋白的上清液。 </li><li>离心夫格提取物以15,000×g离心20分钟，在4℃，将含有蛋白的上清液，注意不要扰乱颗粒。弃沉淀。 </li><li>部分纯化TrCel5A孵育含蛋白质的上清液在55℃下10分钟。允许休息在室温下再保持10分钟，然后离心，15,000 xg下在RT下10分钟。重复部分纯化过程非渗透的植物来获得对照样品。使用这些样本为所有下面的实验。注:蛋白质溶液可以贮存于4℃下长达1周或在-20℃下长达3个月。 </li><li>重复该部分纯化过程非渗透的植物来获得对照样品。使用这些样本为所有下面的实验。注:蛋白质溶液可以贮存于4℃下长达1周或在-20℃下长达3个月。 </li></ol> 5，SDS-PAGE电泳，免疫印迹和TrCel5A-mCherry的&lt;中偶氮CMC酶谱/ P&gt; <ol><li>购买或准备12％（W / V）SDS-PAGE凝胶按照确定的方法15。 </li><li> CMC溶解在水中，得到1.5％（重量/体积）溶液。使CMC-SDS-PAGE凝胶，替换1毫升1.5％CMC的在10ml的SDS-PAGE凝胶的混合溶液中加入1ml水，造成了0.1％（重量/体积）CMC + 12％（w / v的）SDS-PAGE凝胶组合。通常倒入凝胶。注意:请确保管委会在原有的解决方案是完全由搅拌和加热的组合，以40℃溶解（重复制备凝胶溶液直接前）。 </li><li>通过在SDS存在下煮沸，按照已建立的方法12变性的样品。作为阳性对照，用1:500稀释从T.市售的纤维素酶混合物的里氏木霉 ，如T.里氏木霉 ATCC 26921。作为阴性对照，使用来自非渗透的叶（NTEPs）一个类似的方法制备的蛋白质溶液。 </li><li>进行电泳按正常协议15，例如:用200 V为〜50分钟，停止电泳一旦染料前沿到达凝胶的底部。 </li><li>用0.1％染色1的SDS-PAGE凝胶（重量/体积）考马斯亮蓝和脱色用甲醇/冰醋酸混合物。注意:快速染色和脱色可以通过轻轻加热染色和脱色的解决方案， 即通过使用微波对〜15秒预热（但不沸腾）第一染色，然后脱色溶液，改变在10分钟后的脱色液进行时，或者当冷却，并重复与新的脱色液。 </li><li>执行使用既定协议16,17 1 SDS-PAGE凝胶的印迹。使用下面的抗体:抗His-标签（主）和一个多克隆山羊抗兔抗体Fc区已被缀合至碱性磷酸酶（次要）的多克隆兔抗体。注:二级抗体，应选择使用硝基四唑来启用可视化鎓chloride/5-bromo-4-chloro-3&#39; - indolyphosphate对甲苯胺盐（NBT / BCIP），如所述18。 </li><li>进行凝胶内蛋白复性的0.15％（重量/体积），通过培养该凝胶在1.5％（重量/体积）的β-环糊精，20mM磷酸盐 - 柠檬酸盐缓冲液，pH 6.5，在室温下用CMC的SDS-PAGE凝胶轻轻摇动15分钟19。 </li><li>丢弃该β-环糊精溶液中，并简要用H 2 O洗涤该凝胶在50mM醋酸钠缓冲液（pH 4.8）进行15分钟在室温下孵育。使用复性0.15％CMC的SDS-PAGE凝胶在50℃下温育该凝胶在30mM乙酸钠缓冲液pH 4.8 20分钟进行CMC酶谱</li><li>使用复性为0.15％（重量/体积），通过20分钟在50℃下温育该凝胶在30mM的醋酸钠缓冲液（pH 4.8）的CMC的SDS-PAGE凝胶上进行的CMC酶谱</li><li>用0.1％的染色凝胶10分钟w / v的刚果红溶液和脱色30分钟或直到清晰的条带是使用1个月观察娜CL。用0.3％（V / V）乙酸为更清晰的成像洗凝胶。 </li></ol> TrCel5A 6 4-MUC活性测定<ol><li>制备10mM的4-MUC在50％（V / V）甲醇中的储备溶液，并且在黑暗中储存于RT </li><li>在测定之前立即，制备4-MUC 2个工作溶液，1mM的4-MUC组成和60mM的醋酸钠，pH值4.8。 </li><li>含100微升叶蛋白（带或不带TrCel5A可诱导表达），由T. 100微升市售的纤维素酶混合物样品进行这个测试里氏木霉 1:1,000，纯H 2 O的分别。一式三份地运行每个样品。 </li><li>移液管加入100μl的4-MUC工作液加入到96孔板的单独的孔中，再加入100微升各样品。 </li><li>确定0分钟时间点的4-MU荧光通过荧光光谱法，使用360nm的激发波长和465nm的发射波长。 </li><li>孵育板20分钟，在50℃下，覆盖有粘合剂的盖，以防止蒸发。使反应停止冻结（或通过组合加入100μl0.15M的甘氨酸，pH值为10.0，并加入100μl的样品在一个单独的板）。 </li><li>确定的4-MU荧光通过荧光光谱端点，采用360nm的激发波长和465nm的发射波长。减去从端点（20分钟）的时间点0分，获得荧光值的变化。 </li></ol> TrCel5A 7。偶氮羧甲基纤维素酶活性分析<ol><li>制备1.5％的股票（重量/体积）偶氮-CMC被存储在室温下，并含有18 mM的醋酸锌，0.5M醋酸钠，pH值为5和80％乙醇（V / V），在室温储存沉淀溶液温度。 </li><li>测定前夕，准备偶氮CMC 2X工作液，1％组成（重量/体积）偶氮-CMC（以H 2 O）和60 mM醋酸钠，pH值4.8。 </li><li>结合50μl的样品和50μl的工作ING液1.5 ml离心管。 </li><li>含50微升叶蛋白（带或不带TrCel5A瞬时表达）测试样品;将100μl来自T.市售的纤维素酶混合物的里氏木霉稀释1:1,000;纯H 2 O运行样品和标准，一式三份。 </li><li>孵育样品20分钟，在50℃下停止反应，加入250微升的沉淀溶液。涡大力为10秒每个样品，以确保溶液完全混合。 </li><li>再次孵育的样品在室温下10分钟，然后涡旋。离心样品以1,000×g离心10分钟。除去200μl的各样品上清液至96孔板中，注意不要扰乱颗粒。 </li><li>测定用吸收光谱在590nm的波长的光。 </li></ol> 8。PAHBAH TrCel5A的测定<ol><li>使用打孔切割的滤纸圆（〜5.5毫米直径）。加入100μl60 mM的醋酸钠，pH值4.8，和100微升的样品，无论是含叶蛋白（带或不带TrCel5A可诱导表达）或商业T的里氏木霉纤维素酶的混合物，稀释1:1,000，并孵育用滤纸圈20小时，在50℃与300 rpm振摇。对于培养用滤纸每个样品，也没有培养滤纸作为单独的采样控制重复的解决方案。 </li><li>通过在H 2 O的加入5毫升浓盐酸，总共为100毫升的溶液，并储存在室温下溶解PAHBAH制备330 mM的对羟基苯甲酸酰肼（PAHBAH）溶液A。注意:最好的结果，通过加入盐酸，以PAHBAH浆料， 即 30毫升，该温度下搅拌来帮助溶解使体积为100毫升H 2 O之前的一个较小的体积实现</li><li>制备PAHBAH溶液B，含有柠檬酸50mM的钠，10mM的氯化钙和500mM NaOH和存储在RT。 </li><li> IMMED测定iately之前，请准备1毫升PAHBAH试剂1.5倍的工作解决9毫升缓冲溶液，储存于冰上直至使用（须4小时内使用）。 </li><li>准备样品中耐热0.2或0.5 ml离心管。结合5μl的每种溶液的温育，用滤纸（步骤8.1）45微升H 2 O和100μl的PAHBAH工作溶液。样品对照（不育滤纸样品）应在相同浓度（5微升的样品，45微升H 2 O，加入100μlPAHBAH工作液）下运行。也考验5标准（5微升）介于0和葡萄糖0.2g / L的含，纯H 2 O运行样品和标准，一式三份。 </li><li>在100℃下保温样品进行准确的10分钟，然后冷至室温恰好10分钟。移液管溶液加入到96孔板中，并测定光谱在410纳米的波长。从样品中减去个别样品对照组（不带保温过滤纸），以获得最终值。 </li></ol>

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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#8220;Western+Blotting&#8221;:+Electrophoretic+transfer+of+proteins+from+sodium+dodecyl+sulfate-polyacrylamide+gels+to+unmodified+nitrocellulose+and+radiographic+detection+with+antibody+and+radioiodinated+protein+A.">&#8220;Western Blotting&#8221;: Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.</a> Analytical Biochemistry. 112, 195-203 (1981).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elelctrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets+-+procedure+and+some+applications.">Elelctrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets - procedure and some applications.</a> Proceedings of the National Academy of Sciences of the United States of America. 76, 4350-4354 (1979).</li><li>Blake, M. S., Johnston, K. H., Russell-Jones, G. J., Gotschlich, E. 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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulase+linkers+are+optimized+based+on+domain+type+and+function:+Insights+from+sequence+analysis,+biophysical+measurements,+and+molecular+simulation.">Cellulase linkers are optimized based on domain type and function: Insights from sequence analysis, biophysical measurements, and molecular simulation.</a> PLoS One. 7, (2012).</li><li>Boonvitthya, N., Bozonnet, S., Burapatana, V., O'Donohue, M. 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作者什么都没有透露。

纤维素酶的重组表达是非常关注的一个领域，因为更有效的生物燃料生产系统1的愿望。植物提供了许多可能性，成功的纤维素酶生产，具有的功能，如经济采伐技术和自动水解方法是非常理想的特性进行大规模的生物燃料生产。另外，使用不同的本地化的用于生产蛋白质允许（如果需要），翻译后修饰20。改变的植物，以使纤维素酶的组成型表达，同时提供明显的好处，是其可能...

<html>木质纤维素生物质，其转化为液体燃料的退化已经被设想为一个方法，以减少对化石燃料依赖。在建立经济上可行的生物处理系统中的一个显著障碍是在纤维素和半纤维素1的酶促降解。植物表达系统显示为酶在工业规模上生产的巨大潜力。农业系统收获植物经济和体积都已经建立起来，因为他们已经数千年之久。纤维素酶在植物中的生产是可取的，由于像易于自动水解2，以及用于最大限度地通过增加植物细胞壁1消化率使用较低的酶水平的潜在因素。最后，厂房系统允许重组蛋白的靶向植物细胞的特定区域，并能翻译后修饰酶在有需要时3。 ve_content"&gt; 烟草 （烟草）是很常用的植物模式生物的外源蛋白表达的研究，由于其快速增长和生物量的积累功能4。重组蛋白的瞬时表达是一种技术，它允许在短的时间周期5蛋白的生产，同时保持柔韧性，以定位和由此所选择的蛋白， 即纤维素酶的翻译后修饰。这使得生产的纤维素酶（S）的基本分析，同时也奠定了在植物中进一步表达策略。使用这样的瞬时表达的策略，从糖基水解酶家族5，Cel5A，从真菌宿主里氏木霉 （ 红褐肉座菌 ）衍生的6内切葡聚糖酶的产生（以下简称为TrCel5A）。 TrCel5A是一个42 kDa蛋白被糖化本身，是路政署高度活跃roylzing纤维素链7。 这里所描述的瞬时表达技术是基于一个较为常用的系统上，渗入植物叶与土壤杆菌携带目的基因在适当的表达载体中。以允许成功的在植物中表达的快速分析，TrCel5A也可以被表达为融合蛋白的mCherry，最初来源于Discosoma藻单体荧光蛋白。蛋白DsRed的8，具有一个额外的6组氨酸残基（His-标签）融合到在C-末端（TrCel5A-的mCherry）。异源融合蛋白，TrCel5A-的mCherry，表达因此可以生长的植物中通过使用绿色光检查的mCherry分散监控。如果需要，该植物材料的薄的部分可以根据绿色光被显微镜检查，以确定该蛋白的特异性定位。对于这项工作，信号和转录坐在肽在结构注册成立，本地化的异源蛋白出口到内质网9。 以分析植物中表达的纤维素酶，包括TrCel5A，一些纤维素酶活性测试可以被执行的活动。总可溶性植物蛋白的提取后，TrCel5A可以部分地用热温育技术纯化。蛋白质大小是使用SDS-PAGE随后通过Western印迹法确定。酶谱法可用于分析活性对底物， 例如纤维素这一直是羧基甲基化（使羧甲基纤维素:CMC）为溶解度10。纤维素酶和葡糖苷酶的活性可以通过用β-D-纤维二糖苷相关的荧光团4 - 甲基伞形酮（4-MU）（4-MUC组合）进行监测。评估切葡聚糖酶活性的另一种方法涉及到CMC的光谱测定分析已用的偶氮-D相关联的你们（Remazolbrilliant蓝R）11。此外，这两种内切葡聚糖酶和纤维素酶的范围中的蛋白质的活性可以通过使用糖分析测试，如对羟基苯甲酸酰肼（PAHBAH）测定12进行监测。这些技术可用于澄清和量化感兴趣的表达纤维素酶的活性。

<table><tbody><tr><td>4-methylumbelliferyl &amp;beta;-D-cellobioside</td><td>SIGMA-ALDRICH</td><td>M6018</td><td></td></tr><tr><td>Acetic acid</td><td>ROTH</td><td>3738</td><td></td></tr><tr><td>Acetosyringon (concentrated)</td><td>ROTH</td><td>6003</td><td></td></tr><tr><td>Antibiotic - kanamycin</td><td>ROTH</td><td>T832</td><td></td></tr><tr><td>Antibiotic - rifampicin</td><td>ROTH</td><td>4163</td><td></td></tr><tr><td>Antibiotic - carbenicillin</td><td>ROTH</td><td>6344</td><td></td></tr><tr><td>Antibody - rabbit anti His(6) pAb</td><td>ROCKLAND</td><td>600-401-382</td><td></td></tr><tr><td>Antibody - goat anti rabbit AP, FC pAb</td><td>DIANOVA</td><td>111-055-008</td><td></td></tr><tr><td>Azo-carboxymethyl cellulose</td><td>MEGAZYME</td><td>S-ACMC</td><td></td></tr><tr><td>&amp;beta;-cyclodextrin</td><td>SIGMA-ALDRICH</td><td>C4805</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>SIGMA-ALDRICH</td><td>C5080</td><td></td></tr><tr><td>Carboxymethyl cellulose</td><td>ROTH</td><td>3333.1</td><td></td></tr><tr><td>Cellulase from &lt;em&gt;Trichoderma reesei&lt;/em&gt; ATCC 26921</td><td>SIGMA-ALDRICH</td><td>C2730</td><td></td></tr><tr><td>Congo red</td><td>SIGMA-ALDRICH</td><td>75768</td><td></td></tr><tr><td>Coomasie brilliant blue G 250</td><td>ROTH</td><td>9598.2</td><td></td></tr><tr><td>D(+)-glucose</td><td>ROTH</td><td>8337</td><td></td></tr><tr><td>Ethanol</td><td>ROTH</td><td>K928</td><td></td></tr><tr><td>Filter paper - Rotilabo blotting paper</td><td>ROTH</td><td>CL67</td><td></td></tr><tr><td>NBT/BCIP</td><td>SIGMA-ALDRICH</td><td>72091</td><td></td></tr><tr><td>MES buffer</td><td>ROTH</td><td>4256</td><td></td></tr><tr><td>Methanol</td><td>ROTH</td><td>8388</td><td></td></tr><tr><td>Sodium citrate</td><td>ROTH</td><td>3580</td><td></td></tr><tr><td>Sodium dodecylsulfate</td><td>SERVA</td><td>20765</td><td></td></tr><tr><td>Sodium hydroxide</td><td>ROTH</td><td>6771</td><td></td></tr><tr><td>Soil, Einheitserde classic&amp;nbsp;</td><td>PATZER GMBH</td><td></td><td></td></tr><tr><td>Spectrometer - Infinite M200</td><td>TECAN</td><td></td><td></td></tr><tr><td>Sucrose</td><td>SIGMA-ALDRICH</td><td>S-5390</td><td></td></tr><tr><td>4-Hydroxy benzhydrazide&amp;nbsp;</td><td>SIGMA-ALDRICH</td><td>H9882</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>ROTH</td><td>1058</td><td></td></tr><tr><td>UV light source - BLAK RAY</td><td>UVP</td><td></td><td></td></tr><tr><td>Vibratome - VT1000S</td><td>Leica</td><td></td><td></td></tr></tbody></table>

expression of recombinant cellulase cel5a from trichoderma reesei in tobacco plants

纤维素降解酶，纤维素酶，是研究和产业利益的目标。这些酶在难以培养微生物，如菌丝建设真菌和厌氧菌的优势，加快了在这一领域的使用重组技术。植物表达的方法是理想的系统，用于大规模生产的酶和其它工业上有用的蛋白质。本文中，对于真菌内切葡聚糖酶， 里氏木霉 Cel5A，在烟草中的瞬时表达方法被证实。成功的蛋白表达被示出，使用的mCherry-酶融合蛋白的荧光监测。此外，一组基本的测试是用来检查瞬时表达T的活动里氏木霉 Cel5A，包括SDS-PAGE，Western印迹，酶谱，以及荧光和基于染料的基质降解试验。这里所描述的系统可以被用来产生活性细胞ulase在很短的时间周期，从而通过组成型或诱导型表达系统，以评估植物中用于进一步的生产潜力。

TrCel5A和TrCel5A-的mCherry采用的瞬时表达系统（ 图1A和1B）中成功表达在烟草植物中。检查TrCel5A-的mCherry融合蛋白的表达模式的揭示健康的叶子（ 图1C），这下绿光（ 图1D）显示广泛的蛋白表达。 对烟草植物的叶子有包含被执行的总可溶性蛋白质的提取的瞬时表达TrCel5A蛋白和SDS-PAGE显示了大量的各种蛋白质的人出席（ 图2A...

Watch this Scientific Journal Video about Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants at JoVE.com

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

烟草植物被用于生产真菌纤维素酶，TrCel5A，通过一个瞬时表达系统。的表达可以用荧光融合蛋白进行监测，并且该蛋白质的活性进行了表征后表达。

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 ...

expression-recombinant-cellulase-cel5a-from-trichoderma-reesei

Universidad de Cartagena UDEC

Research

JoVE Journal

Environment

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

12.8K 次观看.  RWTH Aachen University. 烟草植物被用于生产真菌纤维素酶，TrCel5A，通过一个瞬时表达系统。的表达可以用荧光融合蛋白进行监测，并且该蛋白质的活性进行了表征后表达。 

视频: Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

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

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

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The baculovirus-mediated insect cell expression system is one of the systems used to express recombinant eukaryotic secretory proteins with some post-translational modifications. The secretory proteins need to be routed through the secretory pathways for protein glycosylation, disulfide bonds formation, and other post-translational modifications. To improve the existing insect cell expression of secretory plant proteins, a baculovirus expression vector is modified by the addition of either a GP67 or a hemolin signal peptide sequence between the promoter and multiple-cloning sites. This newly designed modified vector system successfully produced a high yield of soluble recombinant secreted plant receptor proteins of Arabidopsis thaliana. Two of the expressed plant proteins, the extracellular domains of Arabidopsis TDR and PRK3 plasma membrane receptors, were crystallized for X-ray crystallographic studies. The modified vector system is an improved tool that can potentially be used for the expression of recombinant secretory proteins in the animal kingdom as well.

Here, we present the protocols for utilizing the insect cell and baculovirus protein expression system to produce large quantities of plant secreted proteins for protein crystallization. A baculovirus expression vector has been modified with either GP67 or insect hemolin signal peptide for plant protein secretion expression in insect cells.

改进杆状病毒表达载体生产昆虫细胞分泌植物蛋白

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The ...

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

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

一种高效的植物多嵌合体荧光融合蛋白的协同表达

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

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 ...

重组纤维素酶Cel5A从表达

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