Name: apoplast-extraction based method to improve the purity of plant produced recombinant protein
Uploaded: 2024-07-05T14:20:00.000+00:00
Duration: 5 min 33 s
Description: Plants are a newly developing eukaryotic expression system being explored to produce therapeutic proteins. Purification of recombinant proteins from ...

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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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+leaf+plasticity+on+the+isolation+of+apoplastic+fluid+from+leaves+of+tartary+buckwheat+plants+grown+in+vivo+and+in+vitro.">The effect of leaf plasticity on the isolation of apoplastic fluid from leaves of tartary buckwheat plants grown in vivo and in vitro.</a> Plants. 12 (23), 4048 (2023).</li><li>Ekanayake, G., Gohmann, R., Mackey, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+quantitation+of+apoplast+hydration+in+Arabidopsis+leaves+reveals+water-soaking+activity+of+effectors+of+Pseudomonas+syringae+during+biotrophy.">A method for quantitation of apoplast hydration in Arabidopsis leaves reveals water-soaking activity of effectors of Pseudomonas syringae during biotrophy.</a> Sci Rep. 12 (1), 18363 (2022).</li><li>Preston, G. 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><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>&amp;beta;-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。

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...

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

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。

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 ...

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

914 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

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2在 1: 一步亲和纯化为蛋白质蛋白质和蛋白质代谢物的平行的分析

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

科研

生物化学

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

哺乳动物 Bestrophin 离子通道的表达与纯化

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as well as synthesis of essential metabolites. These organelles consist of the following three key sub-compartments. The envelope, characterized by two membranes, surrounds the organelle and controls the communication of the plastid with other cell compartments. The stroma is the soluble phase of the chloroplast and the main site where carbon dioxide is converted into carbohydrates. The thylakoid membrane is the internal membrane network consisting of grana (flat compressed sacs) and lamellae (less dense structures), where oxygenic photosynthesis takes place. The present protocol describes step by step procedures required for the purification, using differential centrifugations and Percoll gradients, of intact chloroplasts from Arabidopsis, and their fractionation, using sucrose gradients, in three sub-compartments (i.e., envelope, stroma, and thylakoids). This protocol also provides instructions on how to assess the purity of these fractions using markers associated to the various chloroplast sub-compartments. The method described here is valuable for subplastidial localization of proteins using immunoblotting, but also for subcellular and subplastidial proteomics and other studies.

Here, we describe a method to purify intact chloroplasts from Arabidopsis leaves and their three main sub-compartments (envelope, stroma, and thylakoids), using a combination of differential centrifugations, continuous Percoll gradients, and discontinuous sucrose gradients. The method is valuable for subplastidial and subcellular localization of proteins by immunoblotting and proteomics.

拟南芥叶绿体子隔层的制备及免疫印迹蛋白质组化分析

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as ...

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall process costs. Alternative, cost-effective capture steps are therefore valuable for industrial-scale manufacturing, where large quantities of a single mAb are produced. Here we present a method for the immobilization of a DsRed-based epitope ligand to a cross-linked agarose resin allowing the selective capture of the HIV-neutralizing antibody 2F5 from crude plant extracts without using Protein A. The linear epitope ELDKWA was first genetically fused to the fluorescent protein DsRed and the fusion protein was expressed in transgenic tobacco (Nicotiana tabacum) plants before purification by immobilized metal-ion affinity chromatography. Furthermore, a method based on activated cross-linked agarose was optimized for high ligand density, efficient coupling and low costs. The pH and buffer composition and the soluble ligand concentration were the most important parameters during the coupling procedure, which was improved using a design-of-experiments approach. The resulting affinity resin was tested for its ability to selectively bind the target mAb in a crude plant extract and the elution buffer was optimized for high mAb recovery, product activity and affinity resin stability. The method can easily be adapted to other antibodies with linear epitopes. The new resins allow gentler elution conditions than Protein A and could also reduce the costs of an initial capture step for mAb production.

In this procedure, a DsRed-based epitope ligand is immobilized to produce a highly selective affinity resin for the capture of monoclonal antibodies from crude plant extracts or cell culture supernatants, as an alternative to Protein A.

活联甘蔗，用于亲和色谱树脂的快速发展 - 抗体捕获作为案例研究

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...

Recombinant Production of Bifidobacterial Endoglycosidases for N-glycan Release

Protein glycosylation is a diverse and common post-translational modification that has been associated with many important roles such as protein function, including protein folding, stability, enzymatic protection, and biological recognition. N-glycans attached to glycoproteins (such as lactoferrin, lactadherin, and immunoglobulins) cannot be digested by the host and reach the large intestine, where they are consumed by certain beneficial microbes. Therefore, they are considered next-generation prebiotic compounds that can selectively stimulate the gut microbiome's beneficial microorganisms. However, the isolation of these new classes of prebiotics requires novel enzymes. Here, we describe the recombinant production of novel glycosidases from different Bifidobacteria strains (isolated from infants, rabbits, chicken, and bumblebee) for improved N-glycan isolation from glycoproteins. The method presented in this study includes the following steps: molecular cloning of Bifidobacterial genes by an in vivo recombinational cloning strategy, control of transformation success, protein induction, and protein purification.

Recombinant Production of Bifidobacterial Endoglycosidases for N-glycan Release

Bifidobacteria possess a unique genomic capability for N-glycan cleavage. Recombinantly producing these enzymes would be a promising novel tool to release bioactive N-glycans from glycoprotein-rich substrates such as colostrum.

Protein glycosylation is a diverse and common post-translational modification that has been associated with many important roles such as protein function, ...

Assaying for Inorganic Polyphosphate in Bacteria

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many bacteria. There are a variety of methods for quantifying polyP in biological materials, many of which are either labor-intensive or insensitive, limiting their usefulness. We present here a streamlined method for polyP quantification in bacteria, using a silica membrane column extraction optimized for rapid processing of multiple samples, digestion of polyP with the polyP-specific exopolyphosphatase ScPPX, and detection of the resulting free phosphate with a sensitive ascorbic acid-based colorimetric assay. This procedure is straightforward, inexpensive, and allows reliable polyP quantification in diverse bacterial species. We present representative polyP quantification from the Gram-negative bacterium (Escherichia coli), the Gram-positive lactic acid bacterium (Lactobacillus reuteri), and the mycobacterial species (Mycobacterium smegmatis). We also include a simple protocol for nickel affinity purification of mg quantities of ScPPX, which is not currently commercially available.

We describe a simple method for rapid quantification of inorganic polyphosphate in diverse bacteria, including Gram-negative, Gram-positive, and mycobacterial species.

细菌中无机聚磷酸盐的检测

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many ...

免疫与感染

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 ...

生物学

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 ...

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 ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

高效的哺乳动物细胞表达和细胞外糖蛋白的结晶一步纯化

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

基于质外体提取的方法提高植物产生的重组蛋白纯度

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此视频中的章节

2在 1: 一步亲和纯化为蛋白质蛋白质和蛋白质代谢物的平行的分析

哺乳动物 Bestrophin 离子通道的表达与纯化

拟南芥叶绿体子隔层的制备及免疫印迹蛋白质组化分析

活联甘蔗，用于亲和色谱树脂的快速发展 - 抗体捕获作为案例研究

Recombinant Production of Bifidobacterial Endoglycosidases for N-glycan Release

细菌中无机聚磷酸盐的检测

高通量的亲和标签的重组蛋白的纯化

高级别瞬态表达重组蛋白的植物高效的农杆菌

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

高效的哺乳动物细胞表达和细胞外糖蛋白的结晶一步纯化