这项工作得到了诺里奇研究公园奖学金 (JR), 联合工程和物理科学研究理事会/生物技术和生物科学研究理事会 (BBSRC) 资助 OpenPlant 合成生物学研究中心赠款 (BB/L014130/1) (M.J.S.、艾欧史密斯);约翰 Innes 中心知识交流和商业化赠款 (BB/KEC1740/1);BBSRC 研究所的战略方案赠款 ' 分子从自然 ' (BB/P012523/1) 和约翰 Innes 基金会 (艾欧史密斯)。我们要感谢乔治 Lomonossoff 提供 pEAQ 媒介和安德鲁戴维斯摄影。

注:在遵循本协议之前, 熟悉所有使用过的物质的安全数据, 并采取适当的安全防范措施。这些措施包括但不限于: 在真空下使用玻璃时佩戴安全眼镜, 以及在通风柜中处理干硅胶。还必须对有关使用转基因材料的地方立法进行检查和观察, 包括遵循适当的遏制程序。
1. 生成一株用于浸润的农杆菌菌株
注:我们在此提供了一个简化的描述, 用于生成合适的. 这些步骤的进一步细节由塞恩斯等人描述。8。
<ol><li>通过 PCR 或合成感兴趣基因的蛋白质编码区, 由 5 ' 和 3 ' attB 序列, 适合于特定于站点的重组克隆协议18,19, 用于生成条目克隆。<ol>
<li>使用前底漆: GGGGACAAGTTTGTACAAAAAAGCAGGCTTANNNNNNNNNNNNNNNNNN, 反向底漆: GGGGACCACTTTGTACAAGAAAGCTGGGTANNNNNNNNNNNNNNNNNN (NNNN... = 序列特定序列)。</li>
		<li>使用 (代表性) PCR 条件: 反应混合物 (25 µL, 总计), 缓冲器 (5x, 5 µL, 见材料表), dNTPs (10 毫米, 0.5 µL), 正向底漆 (10 µM, 1.25 µL), 反向底漆 (10 µM, 1.25 µL), 模板 DNA (可变浓度, 50-250 ng, 0.5 µL), DNA 聚合酶 (2000 U/毫升, 0.25 µL, 见材料表), 无核酸酶水 (到25µL)。运行 thermocycler 如下: 初始变性 (98 °c, 三十年代);开始周期 (98 °c, 三十年代; 45-72 °c, 二十年代; 72 °c, 三十年代/kb) 结束周期;35个周期;最后延伸 (72 °c, 10 分钟)。</li>
		<li>遵循标准的站点特定的重组克隆协议18,19 , 使用任何非卡那霉素的入口向量生成一个条目克隆 (我们使用的 pDONR207 是商业上可用的)。 注:在使用生成 pEAQ DEST1 表达式构造之前, 应通过排序确认条目克隆的完整性.pEAQ-DEST1是可利用的要求从 Lomonossoff 实验室在约翰 Innes 中心在英国诺里奇。</li>
		<li>将 pEAQ DEST1 矢量与步骤1.1.3 中生成的表达式克隆隔离, 并在步骤1.3 中使用之前存储在-20 摄氏度。 注:任何商业上可用的方便自旋柱基质粒纯化试剂盒的设计, 从克隆菌株的大肠杆菌是合适的。按照所选质粒纯化试剂盒的具体说明 (见材料表)。</li>
	</ol>
</li>
	<li>为转换准备化学上合格的农杆菌。<ol>
<li>接种选择性磅 (Luria-Bertani 培养基:10 克/升 bacto-胰蛋白胨, 10 克/升氯化钠, 5 克/升酵母萃取物, 琼脂10克/升 pH 值 7.0) 琼脂板 (50 微克/毫升利福平, 100 微克/毫升链霉素), 通过裸奔a. 农杆菌LBA4404 从甘油的股票文化。一夜之间孵化在28°c 在一个常设孵化器。</li>
		<li>接种50毫升选择性液体 LB 介质 (50 微克/毫升利福平, 100 微克/毫升链霉素), 并从步骤1.2.1 的 LBA4404 细胞的样本.孵化过夜在28°c 在一个震动孵化器在200转每分钟。</li>
		<li>冷却的文化从步骤1.2.2 冰10分钟, 然后颗粒的A. 农杆菌细胞离心在4°c 和 4500 x g 5 分钟 (丢弃上清)。</li>
		<li>并用重悬 1.2.3 1 毫升的冰冷20毫米水 CaCl2溶液中的颗粒, 并从步骤1.2.3 重复离心步骤 (丢弃上清)。</li>
		<li>并用重悬从步骤1.2.4 在1毫升冰冷20毫米水 CaCl2溶液中的颗粒, 整除导致悬浮在50µL 批次。闪光冻结的整除数在液体 N2和存储在-80 摄氏度之前使用。</li>
	</ol>
</li>
	<li>用步骤1.2 中生成的隔离 pEAQ-HT-DEST1 向量转换准备好的 LBA4404 .<ol>
<li>解冻50µL 的A.在冰上步骤1.2 产生的农杆菌悬浮, 并孵育 100 \ u2012 200 ng 的孤立的 pEAQ-HT-DEST1 向量, 生成的步骤 1.1, 在0°c 5 分钟。</li>
		<li>冷休克的孵化悬浮从步骤1.3.1 在液体 N2三十年代, 然后允许温暖的室温。</li>
		<li>添加 200 ul 的 soc 介质 (soc:20 克/升 bacto-胰蛋白胨, 5 克/升酵母提取物, 0.58 克/升氯化钠, 0.19 克/升氯化钾, 2.03 克/升氯化镁2, 2.46 克/升硫酸镁7水合物, 3.6 克/升葡萄糖) 到冷休克悬浮从步1.3.2 和孵化为4小时在28°C 在摇晃的孵化器在200转每分钟。</li>
		<li>接种一个选择性 LB 琼脂板 (50 微克/毫升利福平, 50 微克/毫升卡那霉素, 100 微克/毫升链霉素) 与 SOC 文化从步骤1.3.3。彻底传播, 并孵化3天在28°c 在一个常设孵化器。</li>
		<li>接种5毫升选择性 LB 培养基 (50 微克/毫升利福平, 50 微克/毫升卡那霉素, 100 微克/毫升链霉素) 与一个单一的殖民地从步骤1.3.4。孵化过夜在28°c 在一个震动孵化器在200转每分钟。</li>
		<li>添加 200 ul 的甘油到1毫升的液体养殖从步骤 1.3.5, 彻底混合, 并存储在-80 摄氏度。 注:这种文化可以恢复到未来的实验通过裸奔到 LB 琼脂板与适当的抗生素 (如下所述)。</li>
	</ol>
</li>
</ol>2. 准备渗透悬浮液
<ol><li>在步骤1中产生的甘油种群培养基中接种一株选择性的 LB 琼脂板, 其条纹为所需的.一夜之间孵化在28°c 在一个常设孵化器。 注:在 pEAQ-HT-DEST1 向量内携带tHMGR的应变也可能包括在内。<ol>
<li>单独接种50毫升选择性 LB 培养基, 每个 a 的样本. 在步骤2.1 中生长的农杆菌菌株, 并在28摄氏度在一个摇晃的孵化器中以200转每晚孵化。</li>
		<li>从步骤2.1.1 到1000毫升的选择性 LB 培养基 (利福平50微克/毫升, 卡那霉素, 50 微克/毫升链霉素/毫升), 并在100摄氏度的一夜之间, 在一个摇晃的孵化器中单独转移50毫升的培养基 28 rpm。</li>
		<li>通过离心 (4000 x g), 从步骤2.1.2 中产生的液体培养物中分离出 A.</li>
		<li>单独并用重悬从步骤2.1.3 在50毫升新制备的 mma 缓冲器 (mma 缓冲器:10 毫米 2-(n-吗啉代)-ethanesulphonic 酸, pH 5.6 (KOH), 10 毫米氯化镁2, 150 微米 3 ' 5 '-藜 4 '-苯乙酮) 和在室温下孵化黑暗的1小时。</li>
		<li>将每个a的适当体积组合在一起 (在步骤2.1.4 中生成), 以在10升总最终体积内为每种应变产生至少0.2 的最终 OD600 。</li>
		<li>将附加的 MMA 缓冲器添加到步骤2.1.5 中生成的复合悬浮液中, 最终体积为1升 (在步骤3中立即使用)。 注:在渗透过程中, 为保持液位, 最好准备另外2升的渗悬浮液。</li>
		<li>准备1升10x 的强力 MMA 缓冲器。</li>
	</ol>
</li>
</ol>3. 进行真空渗透
注:请参见图 2 , 说明真空渗透装置的结构。使用5周大的benthamiana植物在 9 cm x 9 cm 罐为操作步骤。幼苗应播种在 F1 堆肥 (例如从犁庭扫穴) 和种植2周前被转移到单独的盆含有 F2 堆肥。植物应该生长在25摄氏度的温室中, 每天有16小时的光照 (必要时使用辅助照明), 每天浇水, 最大密度为100株/米2。
<ol><li>将步骤2中生成的1升渗透悬浮液和1升的10x 强力 MMA 缓冲液转移到渗透浴中, 再加8升水。<ol>
<li>删除定制的植物持有人的可拆卸的翼, 插入4棵植物 (5 星期老植物看见前面笔记), 并且重新连接翼。将保持架和放置在渗透浴的顶部, 以便将叶子浸入渗透悬浮液中。 注:确保悬浮液位达到厂架顶面。如果需要, 增加额外的渗透悬浮液的水平。</li>
		<li>将浸入式浴缸与浸没的植物转移到入渗室并关上门。确保吸收上的进气阀门关闭。打开真空泵, 将内联阀打开到真空储罐中, 然后将真空进气阀放进入渗室。</li>
		<li>一旦渗透腔内的压力降低了880毫巴, 关闭真空进气阀, 打开进气阀门。一旦渗透室回到大气压, 打开门, 并删除渗透浴。</li>
		<li>提高植物的持有者, 直到植物不再淹没在渗透悬浮, 然后轻轻摇动持有者允许过剩的悬浮跑掉的叶子回到渗透浴。</li>
		<li>将持票人返回直立位置, 取下可拆卸的机翼, 取出植株。</li>
		<li>重复步骤3.1.1 到 3.1.5, 直到所需的植物数量被渗透。</li>
		<li>蒸压釜使用前的渗透悬浮处理。</li>
		<li>在随后的实验中再利用之前, 先用高压釜进行渗透浴。</li>
		<li>用70% 乙醇清洗并在随后的实验中再利用前离开空气干燥, 对渗透室内部进行消毒。</li>
	</ol>
</li>
</ol>4. 种植渗透植物
<ol><li>在温室中, 以100株/米2的最大密度从步骤3排列渗透植物。</li>
	<li>生长5天在25摄氏度和16小时每天的光 (使用补充照明时, 需要), 和水每天。</li>
</ol>5. 收获被渗透的叶子
<ol><li>经过5天的成长, 收获了树叶。</li>
	<li>蒸压釜废料厂的物料、花盆和土壤处置前。</li>
</ol>6. 干燥收获的叶子
注:下面描述的确切的冻干过程取决于可用的冻干装置的性质。
<ol><li>Lyophilize 收获的叶子, 直到干燥。<ol>
<li>将收获的叶子放在2毫米厚的聚丙烯袋中。将收获的叶子冷冻在-80 摄氏度冷藏库中, 保存30分钟。</li>
		<li>用别针在袋子上打上许多小孔。把袋装的冷冻树叶放在冻干机中央的房间里。确保所有的烧瓶附件水龙头都关闭, 然后打开冻干机。确保冷凝器达到至少-50 摄氏度, 压力降低到至少0.15 毫巴, 然后一夜之间离开。</li>
		<li>关闭冻干机和排气真空通过打开一个烧瓶附件水龙头。从中央腔中取出干燥的叶子, 然后进行步骤7。</li>
	</ol>
</li>
</ol>7. 加压溶剂萃取
注:程序步骤是指使用商业上可用的 PSE 仪器 (见材料表)。有关操作的详细信息, 请参阅制造商的文献。该仪器并行进行4提取。
<ol><li>通过一种方便的方法将干燥的叶子研磨成一粒粉末 (例如,对于多数三萜类化合物, 使用家用食品处理器, 或者在包内手动粉碎叶子)。 注:通常不需要用杵和砂浆在液氮下研磨。<ol>
<li>将叶粉与石英砂 (0.3 \ u2012 0.9 毫米, 20% 按体积) 混合在适当的容器内;这起到了分散剂的作用, 提高了萃取过程的效率。</li>
	</ol>
</li>
	<li>准备4个提取细胞 (120 毫升) 填充。为此, 反转萃取单元并插入玻璃纤维过滤器, 接着是金属熔块和保温插头。将萃取单元返回到直立位置, 并添加1厘米深的石英砂层 (0.3 \ u2012 0.9 毫米)。</li>
	<li>填充抽取单元格。<ol>
<li>用在步骤7.1 中生成的准备好的叶粉填充萃取单元, 直至填充线。如果需要, 在这个过程中轻轻地压缩粉末, 以便在每个萃取单元中增加更多的量。</li>
		<li>添加一小层石英砂 (0.3 \ u2012 0.9 毫米) 到包装粉顶部, 并插入顶部纤维素过滤器。</li>
		<li>将填充的提取单元插入 PSE 仪器并运行所需的方法。使用下列设置和材料;溶剂: 100% 乙酸乙酯;温度: 100 °c, 压力: 130 巴。运行3循环如下: 周期 1: 0 分钟保持时间, 周期2和 3: 5 分钟保持时间, 结束与2分钟溶剂冲洗, 和12分钟氮气冲洗。 注:最好先对小尺度下的萃取方法进行优化。然而, 下面的方法通常是足够的氧化三萜苷元。每个萃取单元的白酒可以通过指定废物线作为提取目标, 而不是标准的单独的收集线, 结合在同一外部收集容器中。注意所用溶剂的用量取决于萃取单元的包装和设定的温度和压力。上述方法通常产生约800毫升提取液4平行萃取。</li>
		<li>重复步骤7.2 至 7.3.3, 直到所有的叶粉被处理。</li>
		<li>将组合萃取液浓缩成干燥, 通过真空旋转蒸发。检查预期产量 (即深绿色泥浆)。 注:具体的程序可能会因设置可用的旋转蒸发器而有所不同。在执行旋转蒸发时, 应始终佩戴眼部防护, 并在真空条件下检查玻璃器皿是否损坏。<ol>
<li>打开冷水机回收器, 并允许热传导液冷却到至少5摄氏度。打开水浴, 将温度设置为35摄氏度。</li>
			<li>将萃取液转移到蒸发瓶中 (不要填满瓶中一半以上的体积)。如果总容积超过这一量, 将酒的批次 (在同一瓶中) 集中。将填充的蒸发烧瓶连接到旋转蒸发器的蒸气管 (用关节夹固定)。</li>
			<li>调整烧瓶的升降位置和角度, 以近似45°角将蒸发瓶的底部第三个浸入水浴中。开始的瓶子旋转的速度, 开始蔓延的萃取液的瓶子的墙壁。</li>
			<li>确保排气水龙头关闭, 并开始减少压力 (使用真空泵的控制器), 直到在冷凝器和接收器之间观察到适度的滴水。 注:不要让萃取液开始沸腾。如果发生这种情况, 减少真空的强度, 使蒸馏得到控制。</li>
			<li>监测和调整真空强度和旋转速度的适当, 直到所有的溶剂被蒸发。</li>
		</ol>
</li>
	</ol>
</li>
</ol>8. 碱性离子交换树脂去除叶绿素的方法
注:以下步骤中引用的数量假定为 100 \ u2012 150 工厂的原始工作规模。步骤8不适用于含有羧酸基团的产品, 或在诸如酯类等基本条件下水解的功能基团。
<ol><li>溶解在步骤7中生成的少量乙醇的粗萃取物。</li>
	<li>添加50毫升的碱性离子交换树脂珠 (见材料表), 以输出的步骤 7.1.1, 并在室温下摇动30分钟。 注:不要用震动来代替搅拌装置的使用。磁性或机械的头顶搅动会损害树脂珠子的完整性。适当的搅拌也可以方便地实现通过缓慢旋转的反应瓶在旋转蒸发器与真空设置在大气压或断开。</li>
	<li>每30分钟添加50毫升碱性离子交换树脂珠, 直到反应完成;碱性离子交换树脂珠的颜色会从粉红色变为绿色, 而液相则会由绿色变为橙色, 或视比例而变暗褐色。<ol>
<li>如果有疑问, 反应已经完成, 样品0.5 毫升的液体。通过硅藻土的一小柱过滤样品, 观察滤液的颜色;反应通常在1.5 小时之内完成。</li>
	</ol>
</li>
	<li>用硅藻土的短柱过滤反应混合物。<ol>
<li>通过真空过滤, 或通过用手波纹管的玻璃闪光色谱柱施加压力来执行此操作。如果过滤速度减慢, 用搅拌棒扰乱硅藻土垫的顶部。收集滤液。</li>
	</ol>
</li>
	<li>用乙醇冲洗所收集的树脂珠子, 后跟1:1 乙醇: 己烷, 最终己烷。使用足够的冲洗量并用重悬在硅藻土柱顶部的珠子。</li>
	<li>将滤液与步骤8.4 相结合, 从步骤8.5 中冲洗出来, 在真空下将其浓缩成干燥。</li>
</ol>9. 初始粗分馏
注:以下方法通常适用于典型氧化三萜苷元, 并采用自动闪光层析仪。有关操作的详细信息, 请参阅制造商的文献。
<ol><li>将步骤8中生成的粗产品吸附到闪光级硅胶 (孔径:60 Å, 粒度:35 \ u2012 75 微米), 用于通过干荷载方法应用于闪光色谱盒。<ol>
<li>将步骤8中生成的原油分解成最小体积的适当有机溶剂。确保完全溶解。 注:二氯甲烷与少量甲醇的加法通常是适当的溶剂选择。</li>
		<li>拧开100克预先填入闪光色谱盒的顶部 (见材料表), 取出插入物以显示干装头空间。使用头部空间来测量合适的硅凝胶体积以进行干荷载。</li>
		<li>将所测的硅胶的测量量转移到原油的溶液中, 然后在真空下通过旋转蒸发去除溶剂。 注: 硅胶应返回自由流动的粉末。在蒸发过程结束时, 可能需要温和的刮擦, 从蒸发瓶的侧面释放硅胶。</li>
		<li>平衡100克闪光色谱盒在100毫升/分钟, 至少5列体积100% 己烷, 但理想的是, 直到墨盒的内容完全和均匀润湿。</li>
		<li>将步进9.1.3 中产生的制备的干加载二氧化硅凝胶转移到平衡100克闪光色谱盒的干装头空间, 通过浇注。悬浮在己烷和宽嘴吸管可以用来帮助转移任何剩余的干加载硅胶。</li>
		<li>用100% 己烷将所转移的干装硅胶床湿润, 从干荷载顶空中除去空气。</li>
		<li>运行以下色谱法: 溶剂 [A]: 己烷, 溶剂 [B]: 醋酸乙酯, 流速: 100 毫升/分, 梯度: 0% [b] 到 100% [b] 3000 毫升, 收集: 收集全部, 分数大小: 250 毫升。</li>
		<li>用薄层色谱法 (TLC)8或气相色谱-质谱法 (gc-ms)8对馏分进行了分析, 并通过真空下的旋转蒸发, 将含有感兴趣化合物的分数进行干燥。</li>
	</ol>
</li>
	<li>进行下游精细净化, 直到达到理想的纯度水平。 注:下游精细提纯完全是复合型的, 不可能提供标准化的步骤 (参见讨论部分)。</li>
</ol>

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作者没有什么可透露的。

高通入真空渗透允许此协议在我们的实验室中经常使用, 用于制备各种下游应用感兴趣的三萜化合物。这里描述的真空渗透装置很容易被复制。增设真空储层是可取的, 以减少拉动必要的真空所需的时间, 但可以简单地将未修改的真空烤箱作为渗透腔。通过添加合适的冷凝器来保护真空泵理论上是明智的, 但在我们的实验室中我们发现这是不必要的。
如果叶片没有完全浸入入渗悬浮液中, 浸润覆盖率就会受损。这一问题的最小化是确保悬浮液的水平达到工厂持有者的顶面, 并且该植物持有者是一个紧密适合渗透浴。图2所示, 在到位时, 植物持有者的顶面被凹入入渗浴。这是通过面板的中间边缘的持有人, 形成锚点与顶部的渗透浴。入渗悬浮液将需要定期增加以维持悬浮液位。这是最好的添加过量渗透悬浮, 以防止逐步减少 OD600的过程中, 大规模的大量渗透。然而, 在我们的手中, 替代水似乎没有明显的影响, 预期产量的准备规模, 虽然这尚未定量调查。从一组渗透悬浮液中可以渗透多少植物仍然是一个开放的问题。我们通常渗透到100和200植物之间的一组渗透悬浮。另外, 在渗滤过程中, 某些土壤浸入悬浮液是正常的。这还未发现对浸润效果有明显的影响。
在收获期间, 最好 (但不是关键的) 收获只有被渗透的叶子 (有些叶子将生长后渗透)。选择性收获防止稀释与非生产性组织, 否则将增加内源杂质的比例相对于化合物的兴趣。这对下游净化的方便性产生了象征性的影响, 并增加了这些过程的规模。当使用 tHMGR 促进前体供应时, 通常观察到坏死表型在浸润后生长期内发育。这是正常的, 实际上是艾滋病选择性收获和下游干燥过程。干叶粉通常可以储存在一个密封的容器中, 在阴凉, 干燥, 黑暗的地方, 但理想的干燥下真空。这取决于感兴趣的化合物的稳定性。如果选择储存在-80 摄氏度冷藏库中, 请多加小心。确保干燥的叶子在一个防水的容器里, 并在从储藏中移走时, 允许这个容器在打开前温暖到室温。如果不这样做, 将导致 rewetting 的叶子, 阻碍下游处理。
本议定书的收获后步骤为说明目的提供, 并帮助那些可能具有有限的实际化学经验的研究人员。它们可以替代其他许多天然产品提取/纯化技术。正如引言中详细介绍的那样, pse 具有许多优点, 但是商用 pse 设备的资本成本可能很高, 而且并不重要。碱性离子交换树脂处理是一种非常方便的方法, 取代传统的皂化后, 液体/液体的划分。然而, 它不适合含有羧酸基团或功能基团的产品, 在基本条件下水解, 如酯类。这些产品预计将保留在碱性离子交换树脂上。但是, 有可能利用此方法进行捕获和释放过程 (此处未记录)。在传统的皂化方法适当的地方, 碱性离子交换树脂是一种方便的替代品。在步骤9中描述的色谱方法被发现对图 3中所描述的化合物推广。然而, 增加极性的化合物可能需要延长100% 乙酸乙酯洗脱期。在步骤9.2 中, 下游精细提纯完全是复合特定的, 也依赖于尺度。重复循环的小规模的闪光色谱优化梯度, 通常足以达到纯度适当的核磁共振分析。在更大的尺度上, 结晶往往是一种方便的选择。在困难的情况下, 制备或半制备高效液相色谱 (HPLC) 可根据所需的分离化合物数量来使用。一些代表性化合物的下游净化过程的例子可以在别处找到8。
目前的协议, 如这里所介绍的, 是经常在我们的实验室使用, 并已证明实用。然而, 对于表达式平台的进一步优化还有很大的余地。我们的实验室正在进行研究, 以探讨进一步的通路工程和蛋白质表达水平的差异控制如何能进一步改善三萜的产量, 同时对宿主细胞进行毒性作用。也有可能调查细胞内运输系统的操纵20,21和酶的方向到不同的细胞隔间22,23, 途径, 可以改善多氧化事件的效率。还可以探讨改变关键细胞器的基本形态的潜在好处。例如, 观察拟南芥HMGR 膜域的过度表达, 诱导植物内质网肥大24;CYP450 活动的关键站点。该协议非常适合于生产毫克到克的三萜类产品, 并可用于研究设置, 以获取化合物的下游研究 (例如,系统的探索结构-活动关系, 并初步调查的药效学和药代动力学性质的铅化合物的临床兴趣)。然而, 该平台是线性和可靠的可伸缩性, 简单地增加了使用的植物数量, 和通过 agroinfiltration 瞬态表达的实用性已证明在工业规模生产的药物蛋白14, 因此, 有可能扩大这个平台, 用于商业生产高价值的萜。另外, 还可以设想生产稳定的转化, 多基因生物合成途径的最终愿望, 允许大规模种植和继续 ' 耕种 ' 转基因benthamiana菌株生产不同的商业价值化合物。

萜是植物天然产物的最大和结构多样的家庭之一。尽管它们具有巨大的多样性, 但所有的三萜天然产物都被认为是来自于相同的线性前体 23-oxidosqualene, 是植物羟戊酸通路的产物。23-oxidosqualene 的环化是由一个称为 oxidosqualene 环化酶 (OSCs) 的酶家族发起和控制的。这个环化步骤代表了第一级的多样化, 有数以百计的不同的三萜脚手架已报告从自然1。这些支架通过剪裁酶, 包括, 但不限于, 细胞色素 p450s (CYP450s)1,2进一步多样化。这种生物合成途径可能导致巨大的复杂性, 有时导致最终的产品, 这是几乎无法辨认的父三萜。例如, 有效杀虫和拒食 limonoid 印楝素的复杂结构被认为是从 tirucallane 类型3的 tetracyclic 三萜中衍生出来的。
许多三萜类衍生的天然产品, 即使是那些相对未修改的母体支架, 已被证明拥有药用相关的生物活性4,5,6,7。然而, 到目前为止, 这种潜力并没有转化成大量的三萜类衍生药物在诊所。这可以说是 (至少部分地) 对这类化合物的实际综合准入有限的结果, 这一问题会扼杀传统药用对结构-活动关系的探索和铅候选者的发展化学工作流。
benthamiana叶中其他植物中三萜类生物合成酶的瞬态表达可以将 23-oxidosqualene 的内源供给转移到新的高附加值三萜产品的生产上 (图 1)。该过程可用于功能表征候选酶和重建重要的自然发生代谢物的生物合成途径。同样, 它也可以利用组合生物合成方法来生产新的三萜类产品, 这一战略可以导致结构相关的类似物的图书馆, 允许系统地探索的结构性-活动关系生物活性铅化合物8,9。
Agroinfiltration 是在benthamiana叶中实现瞬时表达的一种高效、简便的方法。这一过程涉及叶片的浸润和悬浮的一个...这是通过应用压力, 迫使液体通过气孔, 取代空气在细胞间空间, 并取代它与A. 农杆菌悬浮。细菌将各自的 T dna 转移到植物细胞的内部, 导致浸润叶组织中的局部和瞬态蛋白表达。
虽然任何适于生成转基因植物的二进制向量都可以用于瞬时表达, 但我们利用豇豆花叶病毒 (CPMV) 衍生的Hypertranslational (HT) 蛋白表达系统10, 11. 在这个系统中, 感兴趣的基因由 CPMV RNA-2 的未翻译区域 (UTR) 两侧。5 ' UTR 含有修改, 导致蛋白质翻译水平非常高, 不依赖病毒复制12。这一技术已发展成易于快速 (pEAQ) 的二进制向量序列, 其中包括站点特定的重组克隆协议兼容构造 (pEAQ)10,11.大多数 pEAQ 载体还含有一个西红柿浓密特技病毒衍生 P19 沉默抑制基因13内的 T DNA 部分的表达式卡带, 它绕过需要 coinfiltrate 一个单独的 P19-carrying 菌株, 并提供非常高水平蛋白表达在寄主植物细胞10,11。
使用benthamiana作为表达宿主在使用植物生物合成途径时具有特殊的优势。细胞结构内在地支持适当的 mRNA 和蛋白质处理和适当的分裂, 除了拥有必要的共同酵素, 硝酸 (为 CYP450s) 和新陈代谢的前体。碳源是光合作用;因此, 植物可以简单地生长在优质的堆肥, 只需要水, CO2 (从空气) 和阳光作为投入。该平台也非常方便的共同表达的不同的蛋白质组合, 因为这可以实现弃由不同菌株的A. 农杆菌,否定建立大多基因表达的需要磁带。此外, 通过增加实验中使用的植物数量, 该过程可以线性和可靠地扩展。
我们实验室以前的工作已经证明了这个平台的实用性。这包括制备新的萜用于生物活性测定和规模化, 以达到克数量的分离产品。此外, 在 3-羟基、3-methyglutary 辅酶 A 还原酶 (tHMGR) 的 n-端截断、反馈不敏感形式的共同表达下, 多萜类产品的积累可以增加数倍, 一个速率限制上游酶在羟戊酸途径8。
这种制备规模实验的关键是能够方便地放大渗透过程。在一个典型的实验中, 可能需要成百上千的植物来达到分离产物的目标数量。用手浸润单个叶子 (使用不必要的注射器) 在操作上是苛刻的, 而且常常耗费大量的时间, 使得这种方法不切合常规的放大。真空渗透比手渗透具有优势, 因为它不依赖于操作者的技术水平, 并且允许叶片表面更大面积的渗入。这个做法为药物蛋白质的大规模生产使用商业14。本协议采用一种易于复制的真空渗透装置, 可从商用零件中进行构造。这允许同时渗入多达4种植物, 在短时间内为数以百计的植物提供快速和实际的间歇性渗透 (图 2a -2b)。真空渗透装置由形成渗透腔的真空烘箱 (图 2f) 组成。烤箱通过真空蓄水池连接到水泵。这大大减少了在渗透腔内达到所需真空的时间。植物是固定在一个定制的持有人, 并倒置成一个10升不锈钢水浴填补了一个. 农杆菌悬浮 (图 2a -2c)。完全浸泡的空气部分植物是重要的有效渗透。然后将水浴放在渗透腔内 (图 2d -2e), 并将真空应用于从叶间空隙中抽出空气。一旦压力减少了880毫巴 (一个过程大约1分钟) 渗透室被带来快速地回到大气压在 20-三十年代通过打开烤箱入口阀门, 渗透是完成的。
入渗后五天, 植物材料已准备好收割和随后提取和分离所需的产品 (s)。从这一点上来说, 这一过程仅仅是从叶材料中提取和纯化的天然产物之一, 是任何天然产品化学家都熟悉的工作流程。许多不同的方法为最初提取和随后纯化存在15。最适当的选择方法和所使用的确切条件是高度依赖于化合物的特定化学性质的兴趣, 除了可用的技能和/或设备。在本议定书中, 不可能将收获植物叶材料的下游加工过程中的一个完全推广的方法纳入到孤立的产品中, 这一步骤可以盲目地用于任何有意义的三萜产品, 也不会适当的尝试这样做。然而, 本协议将提供我们实验室所使用的基本工作流程的概述, 以及该过程的早期阶段的一些方法, 在我们的经验证明推广大多数含氧苷三萜产品。这包括两种相对少见的技术, 即加压溶剂萃取 (PSE), 以及一种使用强碱性离子交换树脂去除叶绿素的简便异构相法。
PSE 是一种高效的从固体基质中提取小有机分子的技术。萃取是在压力下 (约100-200 巴) 进行的, 其主要优点是能够使用超过萃取溶剂沸点的缓解温度。与其他热溶剂技术 (如简单回流或索索萃取16) 相比, 这可以显著减少所需的溶剂的时间和用量, 以实现彻底提取。商用台式 PSE 仪器是可用的, 利用可互换的萃取细胞, 和自动化的溶剂处理, 加热和监测。这使得这种技术非常方便。它也可以说是更少的危险, 特别是对于那些实践化学经验有限的操作者。
粗叶提取物在回流后的皂化是液体/液体的分离, 是在随后的纯化或分析之前, 大量去除叶绿素的常用技术。然而, 这往往是在业务上要求较大的规模。此外, 检测的界面, 或产品的损失, 由于形成的乳液可能是一个问题。使用强碱性离子交换树脂进行异相水解可以作为一种方便的选择。水解叶绿素的色素部分保持粘附在树脂上, 可以简单地通过过滤去除。本议定书采用了一种预先报告的分析程序17的制备尺度适应, 该方法采用了一种商业上可利用的碱性离子交换树脂。
下面我们描述了一个详细的和快速的协议, 为萜的制备规模生产利用这个基于植物的平台。本协议在我们的实验室中经常使用, 用于为应用这种结构特性的核磁共振 (NMR) 光谱学, 准备数以万计的半毫克的分离三萜产品, 并/或进一步研究功能检测。

<table><tbody><tr><td>GC-MS instrument</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Thermocycler suitable for performing PCR reactions</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Gel electrophoresis apparatus</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Adjustable air displacement micropipettes (Sizes : 1000 &amp;micro;L, 200 &amp;micro;L, 100 &amp;micro;L, 20 &amp;micro;L, 10 &amp;micro;L, and 2 &amp;micro;L )</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Wide top liquid nitrogen carrier (size: 2 L)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Benchtop microcentrifuge (Capacity: 24 x 1.5/2mL, Max. speed: =/&gt; 15000 rpm)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Benchtop centrifuge (Capacity: 20 x 50 mL, Max. speed: =/&gt; 4000 rpm)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Large Floor-standing centrifuge (Capacity: 4 x 1L, Max. speed: =/&gt; 4000 rpm)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Standing incubator</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Shaking incubator (Flask Clamp sizes: 10 mL, 50 mL, 2 L)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Centrifuge bottles (1 L)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Vacuum infiltration apparatus</td><td>bespoke</td><td>n/a</td><td></td></tr><tr><td>9 x 9 cm plant pots</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Freeze drying apparatus (=/&gt; 8 litre condenser capacity)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Buchi SpeedExtractor 914 with 120 mL extraction cells (PSE instrument)</td><td>Buchi</td><td>SpeedExtractor E-914</td><td></td></tr><tr><td>Rotary evaporator apparatus</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Evaporation flasks (Sizes: 100 mL, 500 mL, 1 L, 2 L)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Duran bottles (Sizes: 1 L, 10 L)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Conical flasks (Sizes 100 mL, 2 L)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Spatulas (assorted sizes)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Analytical balance (5 figures)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Balance (2 figures)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Measuring cylinders (10 mL, 100 mL, 1 L)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>A -80 freezer</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Biotage Isolera instrument with 250 mL collection rack and bottles (automated flash chromatography instrument</td><td>Biotage</td><td>ISO-1EW</td><td></td></tr><tr><td>Safety glasses</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Lab coat</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Autoclave</td><td>any</td><td>n/a</td><td></td></tr><tr><td>&lt;strong&gt;Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Micropipette tips (Sizes : 1000 &amp;micro;L, 200 &amp;micro;L, 100 &amp;micro;L, 20 &amp;micro;L, 10 &amp;micro;L, and 2 &amp;micro;L )</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Microcentrifuge tubes (Size: 2 mL)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Centrifuge tubes (Sizes: 15 mL, 50 mL)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Disposable Transfer pipettes (size 2 mL)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Petri dishes (Size: 100 mL)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Biotage&amp;reg; SNAP KP-SIL 100 g flash cartridges</td><td>Biotage</td><td>FSK0-1107-0100</td><td></td></tr><tr><td>HPLC vials (1 mL), vial inserts and caps</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Disposable purple nitrile gloves</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Top filter for E-914, cellulose</td><td>Buchi</td><td>51249</td><td></td></tr><tr><td>Bottom filter for E-916 / 914, glass fiber</td><td>Buchi</td><td>11055932</td><td></td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Quartz sand, 0.3 - 0.9 mm</td><td>Buchi</td><td>37689</td><td></td></tr><tr><td>Celite 545 (diatomaceous earth)</td><td>Simga-Alrich</td><td>22140-1KG-F</td><td></td></tr><tr><td>Silica gel 60 (0.040 -0.0063 mm)</td><td>Merck</td><td>1.09385.5000</td><td></td></tr><tr><td>2-(N-morpholino)-ethanesulphonic acid</td><td>Melford</td><td>B2002</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Simga-Alrich</td><td>208337-100G</td><td></td></tr><tr><td>KOH</td><td>Simga-Alrich</td><td>60377-1KG</td><td></td></tr><tr><td>3&amp;rsquo;5&amp;rsquo;-dimethoxy-4&amp;rsquo;-acetophenone (acetosyringone)</td><td>Simga-Alrich</td><td>D134406-1G</td><td></td></tr><tr><td>Rifampicin</td><td>Alfa Aesar</td><td>J60836</td><td></td></tr><tr><td>Kanamycin sulfate</td><td>Gibco</td><td>11815-032</td><td></td></tr><tr><td>Streptomycin</td><td>Simga-Alrich</td><td>S6501-100G</td><td></td></tr><tr><td>Gentamycin</td><td>MP biomedicals</td><td>190057</td><td></td></tr><tr><td>Agarose LE gel</td><td>Melford</td><td>MB1203</td><td></td></tr><tr><td>LB broth Millar</td><td>Simga-Alrich</td><td>L3522-250G</td><td></td></tr><tr><td>Agar</td><td>Simga-Alrich</td><td>A5306-250G</td><td></td></tr><tr><td>NaCl</td><td>Simga-Alrich</td><td>S5886-500G</td><td></td></tr><tr><td>KCl</td><td>Simga-Alrich</td><td>P9541-500G</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Alrich</td><td>M2773-500G</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Alrich</td><td>G7021-100G</td><td></td></tr><tr><td>Ambersep&amp;reg;&amp;nbsp;900 hydroxide (basic ion exchange resin)</td><td>Sigma-Alrich</td><td>06476-1KG</td><td></td></tr><tr><td>Hexane</td><td>Fisher</td><td>H/0406/PB17</td><td></td></tr><tr><td>Ethyl acetate</td><td>Fisher</td><td>E/0900/PB17</td><td></td></tr><tr><td>Dichloromethane</td><td>Fisher</td><td>D/1852/PB17</td><td></td></tr><tr><td>Methanol</td><td>Fisher</td><td>M/4056/PB17</td><td></td></tr><tr><td>Ethidium bromide</td><td>Sigma-Alrich</td><td>E7637-1G</td><td></td></tr><tr><td>Ethanol</td><td>VWR</td><td>20821-330</td><td></td></tr><tr><td>Milli-Q water</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Levington F1 Compost</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Levington F2 Compost</td><td>any</td><td>n/a</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit (Plasmid purification kit).</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>NEB 5-alpha Competent E. coli</td><td>Supplier New England Biolabs Ltd</td><td>C2987I</td><td></td></tr><tr><td>pEAQ-HT-DEST1 vector</td><td>Lomonossoff laboratory John Innes Center</td><td>n/a</td><td></td></tr><tr><td>pDONR207</td><td>Thermo-Fisher</td><td>pDONR207</td><td></td></tr><tr><td>A. tumefaciens LBA4404</td><td>Thermo-Fisher</td><td>18313015</td><td></td></tr><tr><td>GATEWAY LR CLONASE(tm) II ENZYME MIX (site-specific recombination cloning protocol)</td><td>Thermo-Fisher</td><td>11791020</td><td></td></tr><tr><td>GATEWAY BP CLONASE (tm) II ENZYME MIX (site-specific recombination cloning protocol)</td><td>Thermo-Fisher</td><td>11789020</td><td></td></tr><tr><td>GO TAQ G2 GREEN MASTER MIX</td><td>Promega UK Ltd</td><td>M7822</td><td></td></tr><tr><td>PHUSION HIGH FIDELITY DNA POLYMERASE</td><td>New England Biolabs Ltd</td><td>M0530S</td><td></td></tr><tr><td>dNTP SET 100mm</td><td>Thermo-Fisher</td><td>10297018</td><td></td></tr><tr><td>RNEASY PLANT MINI KIT</td><td>Qiagen Ltd</td><td>74904</td><td></td></tr><tr><td>RQ1 RNASE-FREE DNASE</td><td>Promega UK Ltd</td><td>M6101</td><td></td></tr><tr><td>Invitrogen SuperScript III First-Strand Synthesis System</td><td>Fisher</td><td>10308632</td><td></td></tr><tr><td>Trytone</td><td>Sigma-Alrich</td><td>T7293-250G</td><td></td></tr><tr><td>Nicotiana benthamiana seeds</td><td>any reputable supplier</td><td>n/a</td><td></td></tr></tbody></table>

transient expression in nicotiana benthamiana leaves for triterpene production at a preparative scale

萜是植物天然产物的最大和结构多样的家庭之一。许多三萜衍生物已被证明具有药用相关的生物活性。然而, 到目前为止, 这种潜力并没有转化成大量的三萜类衍生药物在诊所。这可以说是 (至少部分地) 对这类化合物的实际综合准入有限的结果, 这一问题会扼杀传统药用对结构-活动关系的探索和铅候选者的发展化学工作流。尽管它们具有巨大的多样性, 萜都是从一个单一的线性前体, 23-oxidosqualene。benthamiana中生物合成酶的瞬态异源表达可以将 23-oxidosqualene 的内源性供给转移到生产新的高价值三萜产品上, 而这种产物不是由宿主自然产生的。农业渗透是benthamiana中实现瞬时表达的一种有效、简便的方法。这一过程涉及植物叶片的渗透, 并暂停了根癌农杆菌携带的表达结构 (s) 的兴趣。一个附加的...............经过五天的时间, 渗透的叶片材料可以收获和处理, 以提取和分离产生的三萜产品 (s)。这是一个线性和可靠的可伸缩的过程, 只需增加实验中使用的植物数量就可以了。本文描述了一种利用这种基于植物的平台快速制备萜的协议。该协议采用一种易于复制的真空渗透装置, 允许同时渗入多达四种植物, 使数以百计的植物在短时间内大量渗入。

典型的分离产量取决于研究中的酶/酶组合, 靶化合物的化学稳定性, 以及纯化过程的挑战性。我们以前报告的β-amyrin 衍生物的分离出产量从我们的组合生物合成实验范围从0.12 到3.87 毫克每克干燥N. benthamiana叶粉。这些化合物在氧化水平上广泛存在, 包括酶的非自然组合 (图 3)8。本协议经常在我们的实验室中使用, 用于生产以核磁共振光谱学和下游功能检测为结构表征的数以亿毫克的纯化产品。我们还展示了这一平台的制备效用, 通过生产0.8 克三萜脚手架β amyrin 在纯度大于98%。该试验使用了459株植物, 代表了3.3 毫克每克干benthamiana叶粉8的分离产量。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58169/58169fig1.jpg"> 图 1: 示意图摘要.利用benthamiana叶中生物合成酶的瞬态表达过程的示意图表示, 将 23-oxidosqualene 的内源供应转移到新的高附加值三萜产品的制备生产中。<a href="https://www.jove.com/files/ftp_upload/58169/58169fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58169/58169fig2.jpg"> 图 2: 真空吸收施工及工艺. a)定制的植物持有人一翼分离。b) 定制的植物持有者翅膀连接。c) 反转和淹没植物。d) 插入渗透浴。e) 渗透浴在渗透室内到位。f) 完全真空吸收: (1) 真空泵 (2) 从真空泵到真空储层 (3) 真空储层隔离阀 (4) 真空储层与渗滤室 (5) 压力表 (6) 进气阀 (7) 的连接渗透室 (8) 真空储层。g) 在五天后浸润生长后, 有代表性的叶子从一个植物中渗透到绿色荧光蛋白 (GFP) 的表达结构中。<a href="https://www.jove.com/files/ftp_upload/58169/58169fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58169/58169fig3.jpg"> 图 3: 使用本议定书8对β-amyrin 衍生物的制备生产的以前报告的代表性结果.此表由独立的收益率排序。列的条件格式为相对值的颜色指示。红色 = 高, 绿色 = 低 (虽然中间黄色)。<a href="https://www.jove.com/files/ftp_upload/58169/58169fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale at JoVE.com

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

烟草 Benthamiana叶在制备鳞片上的瞬态表达

烟草 benthamiana叶片中生物合成酶的瞬态异源表达可以将 23-oxidosqualene 的内源供给转移到新的高附加值三萜产品的生产中。这里描述了一个详细的协议, 快速 (5 天) 准备规模生产萜和类似物利用这个强大的植物基础平台。

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

transient-expression-nicotiana-benthamiana-leaves-for-triterpene

Research

JoVE Journal

Biochemistry

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

17.1K 次观看.  John Innes Centre. 烟草 benthamiana叶片中生物合成酶的瞬态异源表达可以将 23-oxidosqualene 的内源供给转移到新的高附加值三萜产品的生产中。这里描述了一个详细的协议, 快速 (5 天) 准备规模生产萜和类似物利用这个强大的植物基础平台。

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

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 ...

科研

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 ...

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

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be used to generate different diterpenoid scaffolds. Purified compounds can be used in various downstream applications, such as metabolite structural analyses, enzyme structure-function studies, and in vitro and in planta bioactivity experiments.

Here we present easy to use protocols for producing and purifying diterpenoid metabolites through the combinatorial expression of biosynthetic enzymes in Escherichia coli or Nicotiana benthamiana, followed by chromatographic product purification. The resulting metabolites are suitable for various studies including molecular structure characterization, enzyme functional studies, and bioactivity assays.

一种可定制的酶化生产与纯化天然产物的方法

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological ...

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 ...

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 ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

荧光互补成像检测在烟草烟叶中瞬时测定蛋白质-蛋白质相互作用动力学

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...

Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing need in developing efficient methods for recombinant antibody production. As of 2019, there were more than 70 FDA-approved monoclonal antibodies, and there is exponential growth potential. Despite their promise, limiting factors for widespread use are manufacturing costs and complexity. Potentially, plants offer low-cost, safe, and easily scalable protein manufacturing&#160;strategies. Plants like Nicotiana benthamiana not only can correctly fold and assemble complex mammalian proteins but also can add critical post-translational modifications similar to those offered by mammalian cell cultures. In this work, by using native GFP and an acid-stable variant of green fluorescent protein (GFP) fused to human monoclonal antibodies, we were able to visualize the entire transient antibody expression and purification process from N. benthamiana plants. Depending on the experiment&#39;s purpose, native GFP fusion can ensure easier visualization during the expression phase in the plants, while acid-stable GFP fusion allows for visualization during downstream processing. This scalable and straightforward procedure can be performed by a single researcher to produce milligram quantities of highly pure antibody or antibody fusion proteins in a matter of days using only a few small plants. Such a technique can be extended to the visualization of any type of antibody purification process and potentially many other proteins, both in plant and other expression systems. Moreover, these techniques can benefit virtual instructions and be executed in a teaching laboratory by undergraduate students possessing minimal prior experience with molecular biology techniques, providing a foundation for project-based exploration with real-world applications.

Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana

We describe here a simple method for expression, extraction, and purification of recombinant human IgG fused to GFP in Nicotiana benthamiana. This protocol can be extended to purification and visualization of numerous proteins that utilize column chromatography. Moreover, the protocol is adaptable to the in-person and virtual college teaching laboratory, providing project-based exploration.

在尼科蒂亚娜本 thamiana中瞬态表达IgG融合蛋白的生产

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing ...

本 氏烟草 叶片中 GFP-His 的高效提取和纯化

Apoplast-Extraction Based Method to Improve the Purity of Plant Produced Recombinant Protein

Plants are a newly developing eukaryotic expression system being explored to produce therapeutic proteins. Purification of recombinant proteins from plants is one of the most critical steps in the production process. Typically, proteins were purified from total soluble proteins (TSP), and the presence of miscellaneous intracellular proteins and cytochromes poses challenges for subsequent protein purification steps. Moreover, most therapeutic proteins like antigens and antibodies are secreted to obtain proper glycosylation, and the presence of incompletely modified proteins leads to inconsistent antigen or antibody structures. This work introduces a more effective method to obtain highly purified recombinant proteins from the plant apoplastic space. The recombinant Green fluorescent protein (GFP) is engineered to be secreted into the apoplast of Nicotiana benthamiana and is then extracted using an infiltration-centrifugation method. The GFP-His from the extracted apoplast is then purified by nickel affinity chromatography. In contrast to the traditional methods from TSP, purification from the apoplast produces highly purified recombinant proteins. This represents an important technological improvement for plant production systems.

作者聚焦:优化质外质体蛋白提取，从植物细胞中高效回收重组蛋白

The purification of recombinant proteins from plant systems is usually challenged by plant proteins. This work provides a method to effectively extract and purify a secreted recombinant protein from the apoplast of Nicotiana benthamiana.

基于质外体提取的方法提高植物产生的重组蛋白纯度  

Plants are a newly developing eukaryotic expression system being explored to produce therapeutic proteins. Purification of recombinant proteins from ...

烟草 Benthamiana叶在制备鳞片上的瞬态表达

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