这项工作得到了中国科学院青年创新促进会 （2021084） 和中国科学院微生物研究所三年行动计划重点部署项目支持基金的支持。

1. 本氏烟草植物的制备
<ol><li>将幼苗块放入托盘中，加入 1 升水并浸泡约 2 小时直至完全饱和。</li>
	<li>将 野生型本氏烟草 种子均匀撒在幼苗块上，盖上盖子，发芽 1 周。在光周期为 18 小时、24°C、相对湿度为 45% 的温室中种植 本氏猪笼 草，每 2 周施肥一次。</li>
	<li>种子发芽后，用镊子轻轻去除幼苗，注意不要损坏其根部。在新托盘的中央挖一个小坑，将幼苗放入其中，然后用土壤轻轻覆盖根部。盖上盖子并继续生长。</li>
	<li>生长到幼苗有三片叶子，盖上盖子。当第 4 片 真叶出现时取下盖子。</li>
	<li>在农作物渗透之前，让植物继续生长两周。</li>
</ol>2. 重组 GFP-His 的构建
<ol><li>根据基因序列进行 N . benthamiana 密码子优化和 GFP 和 PR1a （MGFVLFSQLPSFLLVSTLLLFLVISHSCRAG） 的合成。</li>
	<li>使用 Xba I 和 Sac I 酶对 pCAMBIA1300 载体进行双重消化（参见 材料表）。</li>
	<li>使用上游引物 （TTGGAGAGAACGGGGGACTCTAGAATGGGATT TGTTCTCTTTTC） 和下游引物 （TCCTCG CCCTTGCTCACCATGTCGACCCCGGCACGGCAAGA GTGGG）、使用上游引物 （CCCACTCTTGCCGTGCCGGGGTCGACATGGTGAG CAAGGGCGAGGA）和下游引物（GGGGAAATTCGAGCTCTCAGTGGTGATGGTGGTG GTGAGATCTTCCGGACTTGT）。</li>
	<li>使用同源重组，将 PR1a 和 GFP 克隆到 pCAMBIA1300 植物表达载体中。转移到 大肠杆菌中后，选择单个克隆并将其发送给公司进行测序。测序结果与正确质粒的比较是重组质粒 pCAMBIA1300-PR1a-GFP-His，简称 p35s-PR1a-GFP-His。</li>
</ol>3. 本氏烟草中 GFP-His 的生产 
<ol><li>将 1 μL GFP 质粒加入 50 μL 农杆菌 受体细胞 GV3101 中，轻轻混合并加入电极杯中，电击器电击后取出。</li>
	<li>将转化 的根癌农杆菌 涂抹在含有 50 μg/mL 卡那霉素 （Kan） 和 50 μg/mL 利福平 （Rif） 的 LB 固体培养基板上。在 28 °C 下倒置孵育 48 小时。pCAMBIA1300 载体具有抗性，农杆菌具有 rif 抗性;只有正确的 农杆菌 才能在带有 kan 和 rif 的平板上生长。单克隆克隆是阳性转化体，命名为 根癌曲霉 GV3101-p35s-PR1a-GFP-His （AtGPG）。</li>
	<li>用消毒的牙签吸取适量的 AtGPG，并将其接种到 4 mL 含有 Kan 和 Rif 的液体 LB 中。在 28 °C、220-300 rpm 下培养 24 小时。</li>
	<li>将细菌 1：50 传代到新鲜的 LB（50 μg/mL Kan、10 mM 2-（N-吗啉代）乙磺酸 （MES）、20 μM 乙酰丁香酮 （AS））中，并在 28 °C、220-300 rpm 下孵育 10-12 小时。</li>
	<li>通过以 1500 x g 离心 10 分钟收获细菌，并用 1 mL MMA（10 mM MgCl2、10 mM MES pH 5.6、100 μM AS）重悬沉淀。</li>
	<li>使用 MMA 测量 AtGPG 的光密度 （OD） 并将其调整为 OD600=0.8。在室温下将细菌再孵育 2-4 小时。用无针注射器吸取细菌溶液，轻轻按压叶子的背面，并施加压力使溶液渗透到叶子中。每株植物只注射中间的 3-4 片叶子。</li>
	<li>继续在温室中种植 本氏烟草 3-4 天，然后收获叶子用于蛋白质提取。</li>
</ol>4. 观察 GFP 的亚细胞定位
<ol><li>农艺渗透 48 小时后，选择一片平叶，用 6 mm 的打孔器夹住刀片进行打孔。</li>
	<li>将 30% 蔗糖溶液滴在载玻片上，将叶子样品背面朝上放置，并用盖玻片盖住。</li>
	<li>将激发波长设置为 488 nm，并在放大 400 倍后检测 490-550 nm 处的绿色荧光。准备好幻灯片后拍摄第一张照片，10 分钟后拍摄另一张照片。</li>
</ol>5. 从植物中提取 GFP
<ol><li>AWF 提取<ol><li>在农学渗透后 3 天收集表达重组 GFP 的完整新鲜 本氏烟草 叶。温和地处理叶子并保持叶子的完整性，以避免细胞内蛋白质外流。立即使用收集的叶子进行下一步，以避免蛋白质降解。</li>
		<li>用预冷的消毒 ddH2O 冲洗叶片以去除碎屑。</li>
		<li>称取新鲜叶子，将 20 g 叶子（背面朝下）浸入 100 mL 预冷的 100 mM 磷酸盐缓冲液（PB 100 mM Na2HPO4 和 100 mM NaH2PO4 分别配置并以 2.2：1 pH 7.0 的比例混合）在 200 mL 广口烧杯中。用 100 目尼龙细网纱（见 材料表）和塑料板覆盖叶子。 注：预冷可防止蛋白质降解。缓冲液体积可以根据实验规模进行调整。确保所有叶子都浸入水中。</li>
		<li>将烧杯放入真空泵中。施加 0.8 MPa 真空 1 分钟，然后迅速恢复到大气压。</li>
		<li>从缓冲液中取出叶子，用吸水纸轻轻吸干。将叶子卷起来，用网纱包裹，然后将 5 g 叶子放入每个 50 mL 离心管中。为了收集 AWF，将卷起的叶子顶朝上放在管子中，网纱由管帽固定。</li>
		<li>在 4 °C、500 x g 下离心 5 分钟。取出叶子并使用移液管收集管底部的 AWF。每管可获得约 5 mL 的 AWF。 注：为了最大限度地减少细胞内蛋白质的干扰并提取最大量的 AWF 蛋白质，提取可以重复 2 倍至 3 倍，但不能超过 3 倍。</li>
	</ol></li>
	<li>总可溶性蛋白提取<ol><li>将 20 克叶子在液氮中研磨成细粉。以 1：2 的比例添加提取缓冲液（50 mM Tris-HCl pH 7.4、150 mM NaCl、10% 甘油、1% Triton X-100、0.034% β-巯基乙醇（参见 材料表））。</li>
		<li>在 4 °C 的立式混合器中摇动匀浆 30 分钟。 在 4 °C 下以 13,000 x g 离心匀浆 15 分钟。</li>
		<li>将上清液转移到新管中，重复离心 15 分钟。将澄清的上清液转移到另一个试管中。</li>
		<li>通过 0.22 μm 过滤器过滤上清液（参见 材料表）以获得总可溶性蛋白质提取物。</li>
	</ol></li>
</ol>  
6. 使用镍 （Ni） 亲和层析纯化 GFP-His
注意：在 4 °C 下执行所有步骤。
<ol><li>相对于步骤 5.1.6 中的蛋白质提取物体积，以 1：1000 的比例将 Ni sepharose excel 树脂（参见 材料表）添加到试管中。填料体积等于蛋白提取物体积的 1 /1000。</li>
	<li>依次用 10 倍树脂体积的 ddH2O 和 PB （pH 7.0） 洗涤 Ni 树脂 3 次，并去除上清液。</li>
	<li>将蛋白质提取物与 Ni 树脂孵育 2 小时，以允许完全结合。以 500 x g 离心 5 分钟以去除上清液中未结合的蛋白质。用 20 倍树脂体积的 PB 洗涤 3 次。以 500 x g 离心 5 分钟，然后去除上清液。</li>
	<li>通过加入 10x 树脂体积的 250 mM 咪唑（50 mM Tris-HCl pH 7.4、150 mM NaCl、10% 甘油、250 mM 咪唑）洗脱 GFP-His 并收集上清液。</li>
</ol>7. 蛋白质考马斯蓝染色
<ol><li>向 40 μL 蛋白质样品中加入 10 μL 5x SDS 上样缓冲液，煮沸 10 分钟，然后在 110 V 下以 15% SDS-PAGE 运行 60 分钟。</li>
	<li>用考马斯蓝溶液（0.1% R-250、25% 异丙醇、10% 冰醋酸）染色蛋白质凝胶 2 小时。</li>
	<li>倒出溶液，用脱色溶液洗涤 2 小时。观察凝胶;凝胶上的蛋白质将呈蓝色。</li>
</ol>8. 蛋白质的蛋白质 Western 印迹
<ol><li>向 40 μL 蛋白质样品中加入 10 μL 5x SDS 上样缓冲液，煮沸 10 分钟，然后在 110 V 下以 15% SDS-PAGE 运行 60 分钟。</li>
	<li>以 280 mA 的电流将蛋白质转印到 0.45 μm 硝酸纤维素膜上。用 5% 脱脂牛奶封闭膜 1 小时。</li>
	<li>以 1：5000 稀释抗 His 标签抗体，并将膜与抗体孵育 1 小时。使用 tris 缓冲盐水和 tween 20 （TBST） 将膜洗涤 4 次，每次 5 分钟。</li>
	<li>以 1：10000 稀释抗小鼠抗体，并将膜与抗体孵育 1 小时。使用 TBST 将膜洗涤 4 次，每次 5 分钟。</li>
	<li>以 1：1 的比例混合显影剂（参见 材料表）。向膜中加入 400 μL，反应 1 分钟。使用可视化器曝光 1 分钟后拍摄照片。</li>
</ol>9. 蛋白浓度测定
<ol><li>将 5 μL 蛋白质添加到 1 mL Bradford 1x 染料试剂中，反应 5 分钟。</li>
	<li>归零后确定 OD595 值，并从标准曲线计算蛋白质浓度。</li>
</ol>10. 蛋白质定量 31
<ol><li>对蛋白质样品和标准品进行蛋白质印迹。</li>
	<li>使用 ImageJ 1.5.0 灰度分析通过与标准品的比率对 GFP 进行定量。</li>
</ol>

本 氏烟草 叶片中 GFP-His 的高效提取和纯化

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M., Fones, H. N., Mccraw, S., Rico, A., O'leary, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+infiltration-centrifugation+technique+for+extraction+of+apoplastic+fluid+from+plant+leaves+using+phaseolus+vulgaris+as+an+example.">The infiltration-centrifugation technique for extraction of apoplastic fluid from plant leaves using phaseolus vulgaris as an example.</a> J Vis Exp. (94), e52113 (2014).</li><li>Nandi, S., 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=Process+development+and+economic+evaluation+of+recombinant+human+lactoferrin+expressed+in+rice+grain.">Process development and economic evaluation of recombinant human lactoferrin expressed in rice grain.</a> Transgenic Res. 14 (3), 237-249 (2005).</li><li>Menkhaus, T., 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=Considerations+for+the+recovery+of+recombinant+proteins+from+plants.">Considerations for the recovery of recombinant proteins from plants.</a> Biotechnol Prog. 20 (4), 1001-1014 (2004).</li><li>Streatfield, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approaches+to+achieve+high-level+heterologous+protein+production+in+plants.">Approaches to achieve high-level heterologous protein production in plants.</a> Plant Biotechnol J. 5 (1), 2-15 (2007).</li><li>Yamamoto, T., 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=Improvement+of+the+transient+expression+system+for+production+of+recombinant+proteins+in+plants.">Improvement of the transient expression system for production of recombinant proteins in plants.</a> Sci Rep. 8 (1), 4755 (2018).</li><li>Zhao, J., 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=Wheat+Apoplast-localized+lipid+transfer+protein+TaLTP3+enhances+defense+responses+against+Puccinia+triticina.">Wheat Apoplast-localized lipid transfer protein TaLTP3 enhances defense responses against Puccinia triticina.</a> Front Plant Sci. 12, 771806 (2021).</li><li>Venkataraman, S., 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=Recent+advances+in+expression+and+purification+strategies+for+plant+made+vaccines.">Recent advances in expression and purification strategies for plant made vaccines.</a> Front Plant Sci. 14, 1273958 (2023).</li><li>Witzel, K., Shahzad, M., Matros, A., Mock, H. P., M&#252;hling, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+extraction+methods+for+apoplastic+proteins+from+maize+leaves.">Comparative evaluation of extraction methods for apoplastic proteins from maize leaves.</a> Plant Meth. 7, 48 (2011).</li></ol>

作者声明没有利益冲突。

近年来，使用植物生产治疗性蛋白质迅速扩大 1,2,3,4,5,6。蛋白质编码基因被克隆到表达载体中，并通过农杆菌渗透递送到植物组织中。植物细胞生产后，蛋白质被纯化用于下游应用。通常，重组蛋白是从 TSP 提取物中纯化的。然而，提取过程中植物?...

如今，各种植物生产的重组治疗性蛋白质正在研究中，包括抗体、疫苗、生物活性蛋白、酶和小多肽 1,2,3,4,5,6。由于其安全性、低成本和快速扩大生产的能力，植物正成为越来越多地用于生产治疗性蛋白质的平台 7,8。植物系统中的蛋白质表达和修饰以及蛋白质纯化是决定植物生物反应器生产率的关键因素 9,10。提高植物生产率和获得高纯度靶蛋白是植物基生产系统面临的核心挑战11,12。
重组蛋白的亚细胞定位和修饰取决于在 N 端编码的特异性信号肽，该肽在基因表达过程中将蛋白质靶向其最终细胞器目的地。重组蛋白根据其独特特性被设计为定位于质外质体、内质网 （ER）、叶绿体、液泡或细胞质13。对于抗原和抗体，首选分泌蛋白。在分泌之前，蛋白质通过 ER 和高尔基体进行正确的折叠和翻译后修饰 14,15,16。
在植物中生产后，必须从植物提取物中纯化重组蛋白。通常，蛋白质是从可溶性植物总提取物中纯化的，这些植物提取物含有丰富的细胞内蛋白质、错误折叠和未修饰的产物以及细胞色素17。为了去除杂质，植物提取物进行广泛的分级，包括核酮糖-1,5-二磷酸羧化酶/加氧酶 （RuBisCO） 特异性亲和色谱、植酸盐沉淀、聚乙二醇沉淀以及温度和 pH值 18,19 的调节。这种杂质去除步骤很费力，并且会影响蛋白质质量。
质膜以外的植物细胞区室被定义为质外体，包括细胞壁、细胞间隙和木质部20。水相通常称为质外体洗涤液 （AWF）。AWF 包含细胞分泌的分子，可调节许多生物进程，如运输、细胞壁代谢和应激反应21,22。与 TSP 相比，AWF 的分子成分不那么复杂。从 AWF 中提取重组蛋白可以避免细胞内蛋白、错误折叠产物和细胞质残留物的污染。简而言之，提取脂质在负压下通过气孔进入叶片，并通过温和离心收集AWF 23。虽然分离 AWF 被广泛用于研究质外体 24,25,26,27 内部的生物学进展，但它尚未在植物性生产系统中得到利用。
提高植物生产率和获得高纯度靶蛋白是植物生产系统面临的核心挑战28。通常，蛋白质是从含有大量细胞内蛋白质、错误折叠和未修饰产物以及细胞色素的总可溶性植物提取物中纯化的29，这些物质的后续纯化需要很高的成本30。使用 AWF 蛋白提取方法提取分泌到质外体中的外源重组蛋白，可有效提高重组蛋白的均一性，降低蛋白纯化成本。在这里，我们使用信号肽 PR1a 使外源蛋白分泌到质外体空间，并介绍了一种从 本氏烟草的 AWF 中纯化重组蛋白的详细方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mesh nylon fine mesh yarn</td>
			<td>Guangzhou Huayu Trading limited company</td>
			<td>hy-230724-2</td>
			<td>For AWF protein extraction</td>
		</tr>
		<tr>
			<td>50 ml centrifuge tubes</td>
			<td>Vazyme</td>
			<td>TCF00150</td>
			<td>For centrifuge</td>
		</tr>
		<tr>
			<td>ClonExpress II one step cloning kit</td>
			<td>Vazyme</td>
			<td>C112-02</td>
			<td>For clone</td>
		</tr>
		<tr>
			<td>FastDigest Sac1</td>
			<td>NEB</td>
			<td>R3156S</td>
			<td>For clone</td>
		</tr>
		<tr>
			<td>FastDigest XbaI</td>
			<td>NEB</td>
			<td>FD0685</td>
			<td>For clone</td>
		</tr>
		<tr>
			<td>ImageJ 1.5.0</td>
			<td>/</td>
			<td>/</td>
			<td>For greyscale analysis</td>
		</tr>
		<tr>
			<td>Large filter</td>
			<td>Thermo Fisher</td>
			<td>NEST</td>
			<td>For filtering</td>
		</tr>
		<tr>
			<td>Laser scanning confocal microscopy</td>
			<td>Leica SP8</td>
			<td></td>
			<td>For subcellular localization</td>
		</tr>
		<tr>
			<td>Ni sepharose excel</td>
			<td>Cytiva</td>
			<td>10339806</td>
			<td>For protein purification</td>
		</tr>
		<tr>
			<td>Precast protein plus gel</td>
			<td>YEASEN</td>
			<td>36246ES10</td>
			<td>For SDS-PAGE</td>
		</tr>
		<tr>
			<td>Small filter</td>
			<td>Nalgene</td>
			<td>1660045</td>
			<td>For filtering</td>
		</tr>
		<tr>
			<td>SuperSignal West Femto Substrate Trial Kit</td>
			<td>Thermo Fisher</td>
			<td>34076</td>
			<td>For western blotting</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>SCR</td>
			<td>30188928</td>
			<td>To configure the extraction buffer</td>
		</tr>
		<tr>
			<td>Type rotaryvang vacuum pump</td>
			<td>Zhejiang taizhouqiujing vacuum pump limited company</td>
			<td>2XZ-2</td>
			<td>For vacuum infiltration</td>
		</tr>
		<tr>
			<td>Vertical mixer</td>
			<td>Haimen Qilinbel Instrument Manufacturing limited company</td>
			<td>BE-1200</td>
			<td>For mixing</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>SIGMA</td>
			<td>BCBK8223V</td>
			<td>To configure the extraction buffer</td>
		</tr>
	</tbody>
</table>

apoplast-extraction based method to improve the purity of plant produced recombinant protein

植物是一种新开发的真核表达系统，正在探索以产生治疗性蛋白质。从植物中纯化重组蛋白是生产过程中最关键的步骤之一。通常，蛋白质是从总可溶性蛋白 （TSP） 中纯化的，而各种细胞内蛋白质和细胞色素的存在对后续的蛋白质纯化步骤提出了挑战。此外，大多数治疗性蛋白质（如抗原和抗体）的分泌都是为了获得适当的糖基化，而未完全修饰的蛋白质的存在会导致抗原或抗体结构不一致。这项工作介绍了一种从植物质外体空间获得高度纯化重组蛋白的更有效方法。重组绿色荧光蛋白 （GFP） 经过工程改造，可分泌到 本氏烟草 的质外体中，然后使用渗透-离心法提取。然后通过镍亲和层析纯化提取的质外体中的 GFP-His。与来自 TSP 的传统方法相比，从质外体中纯化可产生高度纯化的重组蛋白。这是植物生产系统的一项重要技术改进。

Nowadays, various plant-produced recombinant therapeutic proteins are under study, including antibodies, vaccines, bioactive proteins, enzymes, and small polypeptides1,2,3,4,5,6. Plants are becoming an increasingly utilized platform for producing therapeutic proteins due to their safety, low cost, and ability to rapidly scale production7,8. Protein expression and modification in plant systems, along with protein purification, are critical factors determining the productivity of plant bioreactors9,10. Enhancing plant productivity and obtaining high-purity target proteins are core challenges facing plant-based production systems11,12.
The subcellular localization and modification of recombinant proteins depends on the specific signal peptide encoded at the N-terminus, which targets the protein to its final organelle destination during gene expression. Recombinant proteins are designed to localize to the apoplast, endoplasmic reticulum (ER), chloroplast, vacuole, or cytoplasm, based on their unique properties13. For antigens and antibodies, secreted proteins are preferred. Before secretion, proteins progress through ER and Golgi for correct folding and post-translational modifications14,15,16.
After production in plants, recombinant proteins must be purified from plant extracts. Typically, proteins are purified from total soluble plant extracts, which contain abundant intracellular proteins, misfolded and unmodified products, and cytochromes17. To remove impurities, plant extracts undergo extensive fractionation, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)-specific affinity chromatography, phytate precipitation, polyethylene glycol precipitation, and adjustment of temperature and pH18,19. Such impurity-removal steps are laborious and can compromise protein quality.
The plant cell compartment beyond the plasma membrane is defined as the apoplast, encompassing the cell wall, intercellular spaces, and xylem20. The aqueous phase is commonly called apoplast washing fluid (AWF). AWF contains molecules secreted by cells to regulate numerous biological progresses like transport, cell wall metabolism, and stress responses21,22. In contrast to TSP, the molecular constituents of AWF are less complex. Extracting recombinant proteins from AWF may avoid contamination by intracellular proteins, misfolded products, and cytoplasmic remnants. Briefly, extraction lipid enters leaves through the stomata under negative pressure, and the AWF is collected by gentle centrifugation23. While isolation of AWF is widely employed to study the biological progress inside apoplast24,25,26,27, it has not been exploited in plant-based production systems.
Improving plant productivity and obtaining high purity target proteins are central challenges for plant production systems28. Typically, proteins are purified from total soluble plant extracts containing large amounts of intracellular proteins, misfolded and unmodified products, and cytochromes29, which require great costs for subsequent purification30. The use of AWF protein extraction methods for the extraction of exogenous recombinant proteins secreted into apoplast can effectively improve the homogeneity of recombinant proteins and reduce the cost of protein purification. Here, we use a signal peptide PR1a to enable exogenous protein secretion into the apoplast space and introduce a detailed method for recombinant protein purification from the AWF of N. benthamiana.

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

基于质外体提取的方法提高植物产生的重组蛋白纯度  

GFP-His 的靶点是分泌到本氏烟草的质外体空间 将 GFP 克隆到 pCAMBIA1300 质粒中，使用 N 端 PR1a 信号肽进行分泌，使用 C 端 His 标签进行纯化，生成 p35s-PR1a-GFP-His （图 1A）。重组蛋白在本 氏烟草 中瞬时表达，并在农杆菌浸润后 3 天使用抗 His 抗体通过蛋白质印迹法检测 30 kDa 条带 （图 1B）。质浆溶解是...

从植物系统中纯化重组蛋白通常受到植物蛋白的挑战。本研究提供了一种从本氏烟草质外体中有效提取和纯化分泌型重组蛋白的方法。

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

apoplast-extraction-based-method-to-improve-purity-plant-produced

Research

JoVE Journal

Biology

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

750 次观看.  Shandong First Medical University. 我们研究了如何更有效地从植物细胞中提取重组蛋白。我们回答了质质体蛋白提取方法在应用于重组蛋白时是否可行的问题。这种提取方法提取分泌到质外体中的蛋白质，因此它们具有均一性。无需细胞破碎，从而减少了细胞内蛋白质的干扰和随后的纯化成本。我们将重点介绍如何增加质外体空间中重组蛋白的分泌，以进一步提高质外体蛋白提...

视频: 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

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.

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.