我们感谢R. Sun和其他学生陈的实验室所作出的贡献，植物材料的产生。我们也感谢D.格林博士对他的支持本科生科研技术和创新学院（CTI）。这项研究是由美国国立卫生研究院资助U01 AI075549和1R21AI101329陈问：和上证授予CTI亚利桑那州立大学的陈问：部分支持。 ，J. M.登特·柳辛格·乌尔塔多和J. Stahnke是由上证所补助金支持的本科生。

1。植物生长<ol><li>将60泥炭颗粒成传播托盘。加入4升自来水和2小时让泥炭颗粒吸收水分。 </li><li>添加2 N.本生种子使用播种机，到每个泥炭颗粒覆盖托盘用一个透明的塑料球，让他们在一个25°C，湿度84％的环境与16/8小时日/夜循环（ 图1A）发芽。 </li><li>取下球形播种后两周，从托盘排水，杰克的化肥加2升的浓度为1.48克/升（ 图1B）。继续生长的植物，在25°C，湿度50％的环境与16/8小时日/夜循环。供应杰克的肥料，每2天每盘2升。 </li><li>第4周时，泥炭颗粒的植物转移到一个新的的托盘承载六家工厂提供足够的空间进一步增长（ 图1C），直到他们准备好要渗透在6周龄（ 图1D）。 </li></ol> 2。 农杆菌文化准备 2.1注射器入渗的制备<ol><li>数A.根癌农杆菌菌株GV3101菌株包含矢量geminiviral p19的，pREP110，pBYGFP，含有卡那霉素（100微克/毫升）的LB琼脂平板上pBYDsRed的。连续一株每板48小时，在30℃和成长。 </li><li>同样地，条纹和生长在LB琼脂培养基上的菌株GV3101菌株窝藏MagnICON的载体的5&#39;模块（pICH15879），3&#39;模块包含靶基因，绿色荧光蛋白，红色荧光蛋白（PICH-GFP或PICH-DsRed的），和整合酶（pCH14011）板含有羧苄青霉素（100微克/毫升）。 </li><li>从LB琼脂平板上的单菌落，接种菌株GV3101菌株窝藏geminiviral载体分为3毫升YENB媒体（0.75％细菌酵母提取物，0.8％营养肉汤中，用NaOH调节pH值至7.5）+卡那霉素（100微克/毫升）在一个15毫升的圆底培养管，增长在30℃的液体培养基摇床过夜300 rpm的旋转速度。 </li><li>同样，接种和GV3101菌株窝藏MagnICON矢量在YENB媒体+羧苄青霉素（100微克/毫升）中的摇床过夜，在30℃下生长</li><li>测量并记录为每个培养液的OD 600值，用分光光度计，并使用下面的公式计算必要的体积（V 子 ）进行传代培养，在10毫升的YENB媒体与0.025的起始OD 600为一个新的文化与适当的抗生素。 V基板（毫升）=（10毫升）×（0.025）/ OD 600 </li><li>传输V基板（毫升）的过夜培养（10 - V 子 ）每个菌株在含有适当抗生素毫升的YENB媒体。植物新培养过夜，在30℃下在250 rpm的旋转速率的混合器，直到OD 600值在1.7-2.0的范围内。 </li><li>测量和记录OD 600值，每个液体培养和使用下面的公式，计算ulate必要的体积（V INF渗透缓冲液在50毫升）稀释，使最终OD 600为0.12，而每个菌株。 = INF V（毫升）（50毫升）×（0.12）/ OD 600 </li><li>传输V INF（毫升）的离心管中的每一种文化和降速细胞离心12,000 XG 2分钟，取出上清液，然后悬浮细胞10毫升渗透缓冲液（10毫米的MES，pH值5.5; 10毫米硫酸镁4）振荡简要。 </li><li>混合再悬浮pBYGFP + pREP110 + p19的细胞中，旋转的细胞以12,000 xg离心2分钟，共50毫升渗透缓冲液中重悬。此农杆菌组合GFP geminiviral表达。 </li><li>同样，以下组合农杆菌细胞混合，降速，重悬在50毫升的渗透液。它们包括（1）pBYDsRed的+ pREP110 + P19 geminiviral红色荧光蛋白的表达，（2）pBYGFP + pBYDsRed + pREP110+ p19的为geminiviral共表达GFP和DsRed的，（3）pREp110 + p19的作为阴性对照的geminiviral表达，（4）PICH-GFP + pICH15879 + pCH14011为MagnICON GFP表达，（5）PICH-DsRed的+ pICH15879 +中pCH14011为MagnICON红色荧光蛋白的表达，及（6）pICH15879 + pCH14011作为的MagnICON表达的阴性对照。 </li></ol> 2.2真空渗透<ol><li>接种和培养各菌株GV3101菌株含有geminiviral载体或MagnICON的载体到15毫升YENB介质中与适当的抗生素在一个50毫升的高压灭菌的烧瓶中生长过夜，上述注射器浸润。 </li><li>测量并记录为每个培养液的OD 600值，用分光光度计，并使用下面的公式计算必要的体积（V 子 ）的一种新的文化与继代培养的起始OD 600为0.025在250毫升的YENB媒体与适当的抗生素。 V基板 </sub&gt;（毫升）=（250毫升）×（0.025）/ OD 600 </li><li>传输V SUB（毫升）过夜培养（250 - V 子 ），1升量瓶中，用适当的抗生素每一株蒸压的YENB媒体毫升过夜所描述的上述注射器浸润成长的新的文化。 </li><li>测量和记录为每个培养液OD 600值，并使用下面的公式来计算必要的体积（V INF）在3升的渗透缓冲液稀释，使最终OD 600为0.12，而每个菌株。 INF V（毫升）=（3000毫升）×（0.12）/ OD 600 </li><li>颗粒和重悬V INF（毫升）各培养中所描述的注射器浸润和渗透缓冲液混合再悬浮培养的按照上述注射器浸润组合获得。 Repellet混合农杆菌细胞和悬浮在3 L的渗透液。 </li></ol> 3.1注射器渗透<ol><li>选择4个6周龄N. benthamiana植物叶5。从每一株植物的顶部算起的第一个三片叶子将会完全A.组合与渗透农杆菌株渗透液。第四片叶子是现货渗透所有四个​​应变组合geminiviral表达或全部三个组合MagnICON表达的。每个工厂将最后一片叶子渗透与阴性对照组的组合。 </li><li>创建一个小缺口，用针在表皮中的叶的背面侧（ 图2A）。注意：确保不要划伤这么难刺穿叶通过双方农杆菌细胞渗透混合物中，将通过穿刺到另一侧的叶。 </li><li>牢牢把握叶的正面，而申请温柔反压的缺口，用一只手的拇指， 农杆菌在渗透缓冲液的混合物注入到缺口中，与无针注射器（ 图2B）。注：由于农杆菌混合物进入叶的间隙，渗透的区域会变成明显的深绿色（ 图2C）。 </li><li>继续农杆菌混合物注入到暗绿色圆圈的缺口，直到停下来扩大。创建另一个缺口，并重复步骤3.1.2-3.1.4直到整个叶片渗透和全叶前三树叶和最后一片叶子变成深绿色。 </li><li>对于第四每一株植物的叶，所有四个（geminiviral矢量）或三个（矢量MagnICON）的组合将被渗入。 农杆菌菌株的每一种组合的车型一个缺口，并且渗透与组合每个缺口。 </li><li>浸润后，将植物生长室和监器后2-15天浸润（dpi）的蛋白表达之间。 </li></ol> 3.2真空渗入<ol><li>真空干燥器和转让3 L渗透缓冲液农杆菌菌株浴缸放置一个浴缸。将到VACUUBRAND隔膜真空泵（ 图3A）的干燥器中。 </li><li>将植物倒挂干燥器板（ 图3B）和降低板的工厂被淹没，直到整个叶和茎系统渗透缓冲板搁在浴缸之上。将干燥器O形圈沿边缘和上腔放干燥器的盖子（ 图3C）。 </li><li>打开真空泵，并开始计时，当真空度达到100毫巴。慢慢打开释放阀，干燥器在100毫巴1分钟后，，允许进入农杆菌成沉水植物组织间隙。重复此步骤的其他onal时间，以确保良好的浸润性。 </li><li>后浸润，使工厂的干燥器中，把它恢复到直立位置。搬厂的增长空间和2-15 dpi的显示器之间的蛋白表达。 </li></ol> 4。荧光蛋白的检测及摄影<ol><li>从2 DPI，搬厂到黑暗的房间，渗透叶子的背面用手提式UV灯UV光照耀。 </li><li>观察红色荧光蛋白GFP或红色荧光的绿色荧光。 GFP散发出更强的信号与长波紫外线光，而红色荧光蛋白是强短波紫外线光下。 </li><li>一个普通的数码相机，没有闪光灯，拍照荧光叶。 </li><li>搬厂后观察或摄影的增长空间。 </li></ol>

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作者没有竞争经济利益。

全球蛋白质为基础的药品的日益增加的需求所需要的新的生产平台，健壮，可伸缩的，低的成本和安全。植物已显示出是一个最有前途的替代生产系统生产药用蛋白。近年来，开发的解构的病毒为基础的载体已启动，瞬时表达的蛋白质在植物中，从而大大提高了植物表达系统2,10的速度和产率。为了进一步优化瞬时表达系统的效用，我们展示了一个简单而有效的和可扩展的方式引入到植物?...

自20世纪70年代以来，植物已被探讨，作为替代品的哺乳动物，昆虫，细菌的细胞培养物的商业生产重组蛋白和蛋白质疗法1。以植物为基础的系统，生物制药的表达，表明承诺在最近几年一些新的治疗方法的疾病，如高雪氏病，H5N1禽流感3，在临床试验中显示了成功。主管机制的开发重组蛋白在植物中表达的几十年中，因为这些最初的实验中已经创建了用于植物为基础的系统中，有三个主要理由改变目前的蛋白质生产范式的潜力。首先，有一个显着的成本下降所致哺乳动物，昆虫，细菌的生物反应器需要相当大的启动成本，昂贵的生长介质，下游纯化的复杂工序4。创造稳定的转基因植物线也可以让他们超越其他表达系统的可扩展性蛋白表达植物可以种植和收获的农业规模5。其次，以植物为基础的表达系统中显着地减少从对人类的蛋白质表达的主机发送的人或动物的病原体的危险性，展示在公众安全6的优越性。最后，植物利用类似于哺乳动物细胞的真核细胞内膜系统，可以进行适当的翻译后修饰的蛋白质，包括糖基化和组件的多个亚单位蛋白质7。这种能力使植物为基础的系统，提前基于原核系统中，如细 ​​菌，因为一个更广泛的重组蛋白类药物，包括单克隆抗体（mAbs），有一个更复杂的结构，并需要大量的翻译后修饰或组件8。 有两个主要的适当疼痛在植物中表达的重组蛋白。第一个是一个稳定的转基因品系，克隆到编码目标蛋白的DNA的表达盒引入到无论是核或叶绿体基因组的发展​​。这样做，外源DNA变成通过遗传后代，可以极大地提高了可扩展性，远远超出其他表达系统1。核基因组的外源DNA的通常是由根癌土壤杆菌感染植物组织实现，或较少，通过微粒轰击的组织9。然后，使用植物激素诱导分化和生长的转基因植物组织如根和叶。无法实现的叶绿体基因组中的变换可以与甲农杆菌 ，但完全依赖于黄金或钨颗粒涂发射弹道与DNA导入植物细胞。表达recombina的第二种方法nt的蛋白质在植物中的瞬时表达通过10。在这种情况下，感兴趣的基因的病毒衍生的载体传递通过A。经历一个过程的充分开发植物叫农杆菌农杆菌。相反交付的基因构建体整合到植物基因组，然后将开始，直接瞬态生产所需的蛋白质，它可以被收获和一个短的潜伏期后，分离。瞬时基因表达提供了更大的整体蛋白的积累，以及一个改进的时间生产蛋白质的优势，如植物准备收获后约1-2周农杆菌11。这是明显快于稳定的转基因植物线，这可能需要数个月至一年的生成，选择和确认的过程。然而，这也是瞬时表达系统的局限性，因为它不会产生遗传稳定的工厂里未列名可以用来产生大规模的商业生产的种子银行。尽管这样的方法已被开发以提高大规模的瞬时表达。在这里，我们演示了一种瞬态蛋白表达烟草植物由A.交付使用解构病毒载体的代农杆菌 。 A.交付正在开发的两个主要方法农杆菌进入植物组织：台秤通过注射器通过真空室的大规模渗透浸润。这里描述的这两个协议都使用N.本生，这是密切相关的普通烟草厂，寄主植物两个荧光蛋白的瞬时表达绿色荧光蛋白（GFP）的，从水母维多利亚和红色荧光蛋白从Discosoma珊瑚（红色荧光蛋白）12,13。 N.本生是最常见的寄主植物重组蛋白，因为它是服从遗传转化，可以产生大量的生物质迅速，是一位多产的种子生产，规模化生产14。的另一个优点，使用全本生作为蛋白表达的主机是可用的多种表达载体2,5。在这项研究中，两个解构病毒载体的烟草花叶病毒（TMV）RNA复制系统（MagnICON向量）和豆黄矮病毒（BeYDV）DNA复制系统（geminiviral向量）4,11是来自于其他的，基于15-18，都是用来携带绿色荧光蛋白和红色荧光蛋白基因，并把他们交N. A.本生细胞通过根癌农杆菌的三DNA构建将被使用的GFP或红色荧光蛋白与MagnICON载体的表达。它们包括的5&#39;模块（pICH15879）含有启动子和其他的遗传元件的驱动表达的靶基因，含有感兴趣的基因的3&#39;模块（PICH-GFP或PICH红色荧光蛋白），以及整合模块（pICH14011）编码一种酶，它集成了5&#39;和3&#39;的模块组合在一起表达后8,15。 3个DNA构建体，还需要表达用geminiviral载体。除了 ​​含有的的目标的基因（pBYGFP或pBYDsRed）的复制的载体，载体编码复制蛋白（pREP110）的扩增所需的目标复制11,14,16。此外，列入番茄丛矮病毒的载体沉默抑制p19的是理想的高层次目标基因的表达11,16。 一般而言，有三种主要的步骤引入到植物细胞的基因重组蛋白通过农杆菌包括植物生长，A.根癌土壤杆菌培养制备，渗透。作为此过程的最终成功的每一步都是关键，因此，详细描述每个两个注射器渗透和下面的真空渗透。

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		 </tr>
		 <tr>
			<td>GFP </td>
			<td>Invitrogen</td>
			<td>V353-20</td>
			<td><a href="http://www.invitrogen.com/" target="_blank">www.invitrogen.com</a> See reference: Lico and Chen, et al 2008</td>
		 </tr>
		 <tr>
			<td>DsRed</td>
			<td>Clontech</td>
			<td>632152</td>
			<td><a href="http://www.clontech.com/" target="_blank">www.clontech.com</a> See reference: Baird and Zacharias, et al 2000</td>
		 </tr>
		 <tr>
			<td>MagnICON Vector</td>
			<td>Icon Genetics</td>
			<td>n/a</td>
			<td><a href="http://www.icongenetics.com/" target="_blank">www.icongenetics.com</a> See reference: giritch and Marillonnet, et al 2006</td>
		 </tr>
		 <tr>
			<td>Geminiviral Vector</td>
			<td>Author's Lab</td>
			<td>n/a</td>
			<td>See reference: Chen and He, et al 2011</td>
		 </tr>
		 <tr>
			<td>N. benthamiana</td>
			<td>Author's Lab</td>
			<td>n/a</td>
			<td><a href="http://www.herbalistics.com.au" target="_blank">herbalistics.com.au</a></td>
		 </tr>
		 <tr>
			<td>Agrobacterium tumefaciens strain gv3101</td>
			<td>Author's Lab</td>
			<td>n/a</td>
			<td>See reference: Lai and Chen 2012</td>
		 </tr>
		 <tr>
			<td>LB Agar Carbenicillin-100, plates</td>
			<td>Sigma</td>
			<td>L0418</td>
			<td><a href="http://www.sigmaaldrich.com/" target="_blank">www.sigmaaldrich.com</a></td>
		 </tr>
		 <tr>
			<td>LB Agar Kanamycin-50, plates</td>
			<td>Sigma</td>
			<td>L0543</td>
			<td><a href="http://www.sigmaaldrich.com/" target="_blank">www.sigmaaldrich.com</a></td>
		 </tr>
		 <tr>
			<td>Magnesium sulfate hepa hydrate</td>
			<td>Sigma</td>
			<td>M2773-500 g</td>
			<td><a href="http://www.sigmaaldrich.com/" target="_blank">www.sigmaaldrich.com</a></td>
		 </tr>
		 <tr>
			<td>Bacto-Tryptone</td>
			<td>Fisher</td>
			<td>73049-73-7</td>
			<td><a href="http://www.fishersci.com/" target="_blank">www.fishersci.com</a></td>
		 </tr>
		 <tr>
			<td>Bacto Yeast Extract</td>
			<td>Becton, Dickinson &amp; CO.</td>
			<td>REF 212750</td>
			<td><a href="http://www.bd.com/" target="_blank">www.bd.com</a></td>
		 </tr>
		 <tr>
			<td>Difco Nutrient Broth</td>
			<td>Becton, Dickinson &amp; CO.</td>
			<td>REF 234000</td>
			<td><a href="http://www.bd.com/" target="_blank">www.bd.com</a></td>
		 </tr>
		 <tr>
			<td>MES hydrate Buffer</td>
			<td>Sigma</td>
			<td>M8250-1kg</td>
			<td><a href="http://www.sigmaaldrich.com/" target="_blank">www.sigmaaldrich.com</a></td>
		 </tr>
		 <tr>
			<td>Carbenicillin</td>
			<td>Sigma</td>
			<td>C1613-1ML</td>
			<td><a href="http://www.sigmaaldrich.com/" target="_blank">www.sigmaaldrich.com</a></td>
		 </tr>
		 <tr>
			<td>Kanamycin</td>
			<td>Sigma</td>
			<td>70560-51-9</td>
			<td><a href="http://www.sigmaaldrich.com/" target="_blank">www.sigmaaldrich.com</a></td>
		 </tr>
		 <tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma</td>
			<td>221465</td>
			<td><a href="http://www.sigmaaldrich.com/" target="_blank">www.sigmaaldrich.com</a></td>
		 </tr>
		 <tr>
			<td>Jack's Fertilizer</td>
			<td>Hummert International</td>
			<td>Jul-25</td>
			<td><a href="http://www.hummert.com/" target="_blank">www.hummert.com</a></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>Vacuubrand MD4 Vacuum Pump</td>
			<td>Fisher</td>
			<td>13-878-113</td>
			<td><a href="http://www.fishersci.com/" target="_blank">www.fishersci.com</a></td>
		 </tr>
		 <tr>
			<td>Vacuum Air Regulator Valve</td>
			<td>Fisher</td>
			<td>NC9386590</td>
			<td><a href="http://www.fishersci.com/" target="_blank">www.fishersci.com</a></td>
		 </tr>
		 <tr>
			<td>Desiccator 12 1/8" with O ring</td>
			<td>Fisher</td>
			<td>08-594-15C </td>
			<td><a href="http://www.fishersci.com/" target="_blank">www.fishersci.com</a></td>
		 </tr>
		 <tr>
			<td>3 L Tub</td>
			<td>Rubber-Maid</td>
			<td>n/a</td>
			<td>Rubbermaid Servn' Saver Bowl, 10-cup will work</td>
		 </tr>
		 <tr>
			<td>plate/shelf 230ML</td>
			<td>Fisher</td>
			<td>NC9489269</td>
			<td><a href="http://www.fishersci.com/" target="_blank">www.fishersci.com</a></td>
		 </tr>
		 <tr>
			<td>Peat Pellet</td>
			<td>Hummert International</td>
			<td>14-2370-1</td>
			<td><a href="http://www.hummert.com/" target="_blank">www.hummert.com</a></td>
		 </tr>
		 <tr>
			<td>Propagation Tray Dome</td>
			<td>hydrofarm</td>
			<td>132052</td>
			<td><a href="http://www.hydroponics.net/" target="_blank">www.hydroponics.net</a></td>
		 </tr>
		 <tr>
			<td>Propagaiton Tray</td>
			<td>hydrofarm</td>
			<td>138758</td>
			<td><a href="http://www.hydroponics.net/" target="_blank">www.hydroponics.net</a></td>
		 </tr>
		 <tr>
			<td>Virbo Hand Seeder</td>
			<td>Gro-Mor INC</td>
			<td>n/a</td>
			<td><a href="http://www.gro-morent.com/" target="_blank">www.gro-morent.com</a></td>
		 </tr>
		 <tr>
			<td>Flora Cart 4 shelf</td>
			<td>Hummert International</td>
			<td>65-6924-1</td>
			<td><a href="http://www.hummert.com/" target="_blank">www.hummert.com</a></td>
		 </tr>
		 <tr>
			<td>15 ml Round Bottom Culture Tubes</td>
			<td>Sigma</td>
			<td>CLS430172-500EA</td>
			<td><a href="http://www.sigmaaldrich.com/" target="_blank">http://www.sigmaaldrich.com</a></td>
		 </tr>
		 <tr>
			<td>Spectrophotometer</td>
			<td>Bio-Rad</td>
			<td>170-2525</td>
			<td><a href="http://www.bio-rad.com/" target="_blank">www.bio-rad.com</a></td>
		 </tr>
		 <tr>
			<td>Spectrophotometer Cuvettes</td>
			<td>Bio-Rad</td>
			<td>223-9950</td>
			<td><a href="http://www.bio-rad.com/" target="_blank">www.bio-rad.com</a></td>
		 </tr>
		 <tr>
			<td>Microcentrifuge Tubes</td>
			<td>USA Scientific</td>
			<td>1415-2500</td>
			<td><a href="http://www.usascientific.com/" target="_blank">www.usascientific.com</a></td>
		 </tr>
		 <tr>
			<td>Benchtop Centrifuge</td>
			<td>Bio-Rad</td>
			<td>166-0602EDU</td>
			<td><a href="http://www.bio-rad.com/" target="_blank">www.bio-rad.com</a></td>
		 </tr>
		 <tr>
			<td>Incubator/Shaker</td>
			<td>Eppendorf</td>
			<td>Excella E25</td>
			<td><a href="http://www.eppendorf.com/" target="_blank">www.eppendorf.com</a></td>
		 </tr>
		 <tr>
			<td>Ultraviolet Light Model#: UVGL-25</td>
			<td>UVP</td>
			<td>95-0021-12</td>
			<td><a href="http://www.uvp.com/" target="_blank">www.uvp.com</a></td>
		 </tr>
		</tbody>
 </table>

efficient agroinfiltration of plants for high-level transient expression of recombinant proteins

哺乳动物细胞培养物是主要的商业化生产人用疫苗和治疗性蛋白质的平台。但是，它不能满足日益增长的全球需求由于其有限的可扩展性和成本高的药品。植物已证明是最有前途的替代，健壮，可伸缩的，低的成本和安全的药品生产平台之一。最近开发的基于病毒的载体，使快速和高层次的重组蛋白在植物中的瞬时表达。为了进一步优化瞬时表达系统的效用，我们展示了一个简单，高效，可扩展的方法引入到植物组织在这项研究中含有目标基因的农杆菌 。我们的研究结果表明，农杆菌与注射器和真空方法所导致的农杆菌介导的高效引入到两个荧光蛋白的叶子和严谨的生产; GFP和DsRed的。此外，我们展示了这两种方法所提供的独特的优势。注射器浸润简单，不需要昂贵的设备。它也允许灵活地渗透到整个假与靶基因，或在一个叶片上的多个目标基因引入。因此，它可以用于实验室规模的重组蛋白的表达，以及用于比较不同的蛋白质或载体的产率或表达动力学。简单的注射器渗透也表明其效用为主题的生物技术在高中和大学教育。与此相反，真空渗入是更强大的，并可以按比例增加的商业生产药用蛋白。它还提供了能够agroinfiltrate植物物种不适合如生菜和拟南芥浸润注射器的优势。总体而言，结合注射器和真空农杆菌的研究人员和教育工作者提供了一个简单，高效和强大的方法瞬态蛋白表达。这将大大促进药物蛋白和发展，促进科学教育。

1。用注射器入渗荧光蛋白的表达为了演示注射器农杆菌浸润到植物组织中的有效性，我们测试了两个荧光蛋白- GFP和DsRed -表达两种不同的解构植物病毒载体- geminiviral和MagnICON -在N本生 。对于N。本生的叶子被完全渗透与农杆菌的含geminiviral的载体，观察GFP的表达，在整个叶面积在紫外光下从早2 dpi的4 DPI（ 图4C）达到峰值积累。这种早期的...

Watch this Scientific Journal Video about Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins at JoVE.com

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

植物提供了一种新的系统，以商业规模生产药物蛋白，更具可扩展性，成本效益和安全比目前的表达范式。在这项研究中，我们报告一个简单方便，且可扩展的方法，引入目标基因<em&gt;农杆菌</em进入植物蛋白的瞬时表达。

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 ...

efficient-agroinfiltration-plants-for-high-level-transient-expression

Research

JoVE Journal

Biology

47.7K 次观看.  Arizona State University. 植物提供了一种新的系统，以商业规模生产药物蛋白，更具可扩展性，成本效益和安全比目前的表达范式。在这项研究中，我们报告一个简单方便，且可扩展的方法，引入目标基因农杆菌

视频: Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

科研

生物学

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system.
As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1].
As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3].
Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4].
The vector used here (pPICZ&alpha;A) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the &alpha;-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector.

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

The protocol describes protein expression using the methylotrophic yeast Pichia pastoris. The preparation of electrocompetent yeast cells, transformation of the vector with the gene of interest into P. pastoris and yeast DNA purification are also performed. Western blot analysis and protein purification build the last steps in this protein expression protocol.

在甲醇酵母表达的重组蛋白毕赤酵母</em

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

高效的生产和纯化重组小鼠Kindlin-3从昆虫细胞生物物理研究

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

高通量定量表达筛选与纯化应用到重组二硫键丰富的蛇毒蛋白产于<em&gt; E。大肠杆菌</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).

In vivo transfection of naked DNA by hydrodynamic gene delivery introduces genes into the tissue of an animal with minimal inflammatory response. Sufficient amounts of gene product are generated such that gene function and regulation as well as protein structure and function can be analyzed.

蛋白通过流体动力基因传递在小鼠体内瞬时表达

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed &#8220;in vitro human cell free expression systems&#8221;. The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 &#181;l of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer&#8217;s Cleft &#8211; 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 &#181;l of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford&#8217;s assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be used for immunizations, immunoassays and protein sequencing.

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Expression of malarial proteins in cell based systems remains challenging. We demonstrate two step and one step IVT (in vitro translation) cell free expression systems for expressing malarial recombinant rhoptry proteins from HeLa cells. We use a Ni-resin affinity based purification system to purify the rhoptry proteins.

基于海拉细胞自由表达系统中的表达<em&gt;疟原虫</em&gt;棒状体蛋白

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

重组人蛋白与HEK293悬浮细胞的瞬时转染高产表达

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

瞬时表达和重组蛋白的细胞定位在培养的昆虫细胞

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...

在植物中：使用根癌农杆菌瞬时转化可可植物

Vacuum-Forced Agroinfiltration for In planta Transformation of Recalcitrant Plants: Cacao as a Case Study

Transient in planta transformation is a fast and cost-effective alternative for plant genetic transformation. Most protocols for in planta transformation rely on the use of Agrobacterium-mediated transformation. However, the protocols currently in use are standardized for small-sized plants due to the physical and economic constraints of submitting large-sized plants to a vacuum treatment. This work presents an effective protocol for localized vacuum-based agroinfiltration customized for large-sized plants. To assess the efficacy of the proposed method, we tested its use in cacao plants, a tropical plant species recalcitrant to genetic transformation. Our protocol allowed applying up to 0.07 MPa vacuum, with repetitions, to a localized aerial part of cacao leaves, making it possible to force the infiltration of Agrobacterium into the intercellular spaces of attached leaves. As a result, we achieved the Agrobacterium-mediated transient in planta transformation of attached cacao leaves expressing for the RUBY reporter system. This is also the first Agrobacterium-mediated in planta transient transformation of cacao. This protocol would allow the application of the vacuum-based agroinfiltration method to other plant species with similar size constraints and open the door for the in planta characterization of genes in recalcitrant woody, large-size species.

作者聚焦：大型木本植物中无组织培养的 农杆菌介导的转化

Here, we present the first protocol for localized vacuum infiltration for in vivo studies of the genetic transformation of large-sized plants. Using this methodology, we achieved for the first time the Agrobacterium-mediated in planta transient transformation of cacao.

真空强制农渗在 植物 内顽固植物转化中的应用——以可可为例

Transient in planta transformation is a fast and cost-effective alternative for plant genetic transformation. Most protocols for in planta transformation ...