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

<ol>
	<li>Zahmanova, G., 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=Green+biologics:+Harnessing+the+power+of+plants+to+produce+pharmaceuticals.">Green biologics: Harnessing the power of plants to produce pharmaceuticals.</a> Int J Mol Sci. 24 (24), 1757 (2023).</li><li>PREVAIL II Writing Group. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+randomized,+controlled+trial+of+ZMapp+for+Ebola+virus+infection.">A randomized, controlled trial of ZMapp for Ebola virus infection.</a> N Engl J Med. 375 (15), 1448-1456 (2016).</li><li>Ma, J. K. C., 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=Regulatory+approval+and+a+first-in-human+phase+I+clinical+trial+of+a+monoclonal+antibody+produced+in+transgenic+tobacco+plants.">Regulatory approval and a first-in-human phase I clinical trial of a monoclonal antibody produced in transgenic tobacco plants.</a> Plant Biotechnol J. 13 (8), 1106-1120 (2015).</li><li>Shanmugaraj, B., Rattanapisit, K., Manopwisedjaroen, S., Thitithanyanont, A., Phoolcharoen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+B38+and+H4+produced+in+Nicotiana+benthamiana+neutralize+SARS-CoV-2+in+vitro.">Monoclonal antibodies B38 and H4 produced in Nicotiana benthamiana neutralize SARS-CoV-2 in vitro.</a> Front Plant Sci. 11, 589995 (2020).</li><li>Takeyama, N., Kiyono, H., Yuki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant-based+vaccines+for+animals+and+humans:+Recent+advances+in+technology+and+clinical+trials.">Plant-based vaccines for animals and humans: Recent advances in technology and clinical trials.</a> Ther Adv Vaccines. 3 (5-6), 139-154 (2015).</li><li>Ward, B. J., S&#233;guin, A., Couillard, J., Tr&#233;panier, S., Landry, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+III:+Randomized+observer-blind+trial+to+evaluate+lot-to-lot+consistency+of+a+new+plant-derived+quadrivalent+virus+like+particle+influenza+vaccine+in+adults+18-49+years+of+age.">Phase III: Randomized observer-blind trial to evaluate lot-to-lot consistency of a new plant-derived quadrivalent virus like particle influenza vaccine in adults 18-49 years of age.</a> Vaccine. 39 (10), 1528-1533 (2021).</li><li>Siriwattananon, K., 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=Plant-produced+receptor-binding+domain+of+SARS-CoV-2+elicits+potent+neutralizing+responses+in+mice+and+non-human+primates.">Plant-produced receptor-binding domain of SARS-CoV-2 elicits potent neutralizing responses in mice and non-human primates.</a> Front Plant Sci. 12, 682953 (2021).</li><li>Leblanc, Z., Waterhouse, P., Bally, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant-based+vaccines:+The+way+ahead.">Plant-based vaccines: The way ahead.</a> Viruses. 13 (1), 5 (2020).</li><li>Sethi, L., Kumari, K., Dey, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+plants+for+efficient+production+of+therapeutics.">Engineering of plants for efficient production of therapeutics.</a> Mol Biotechnol. 63 (12), 1125-1137 (2021).</li><li>Webster, D. E., Thomas, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-translational+modification+of+plant-made+foreign+proteins;+glycosylation+and+beyond.">Post-translational modification of plant-made foreign proteins; glycosylation and beyond.</a> Biotechnol Adv. 30 (2), 410-418 (2012).</li><li>Streatfield, S. J. <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>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>Ullrich, K. K., Hiss, M., Rensing, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Means+to+optimize+protein+expression+in+transgenic+plants.">Means to optimize protein expression in transgenic plants.</a> Curr Opin Biotechnol. 32, 61-67 (2015).</li><li>Su, H., 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=Plant-made+vaccines+against+viral+diseases+in+humans+and+farm+animals.">Plant-made vaccines against viral diseases in humans and farm animals.</a> Front Plant Sci. 14, 1170815 (2023).</li><li>Agrawal, G. K., Jwa, N. S., Lebrun, M. H., Job, D., Rakwal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+secretome:+Unlocking+secrets+of+the+secreted+proteins.">Plant secretome: Unlocking secrets of the secreted proteins.</a> Proteomics. 10 (4), 799-827 (2010).</li><li>Kim, T., Gondr&#233;-Lewis, M. C., Arnaoutova, I., Loh, Y. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dense-core+secretory+granule+biogenesis.">Dense-core secretory granule biogenesis.</a> Physiology. 21, 124-133 (2006).</li><li>Menkhaus, T. J., Bai, Y., Zhang, C., Nikolov, Z. L., Glatz, C. E. <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>Kim, S. T., Cho, K. S., Jang, Y. S., Kang, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+electrophoretic+analysis+of+rice+proteins+by+polyethylene+glycol+fractionation+for+protein+arrays.">Two-dimensional electrophoretic analysis of rice proteins by polyethylene glycol fractionation for protein arrays.</a> Electrophoresis. 22 (10), 2103-2109 (2001).</li><li>Xi, 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=Polyethylene+glycol+fractionation+improved+detection+of+low-abundant+proteins+by+two-dimensional+electrophoresis+analysis+of+plant+proteome.">Polyethylene glycol fractionation improved detection of low-abundant proteins by two-dimensional electrophoresis analysis of plant proteome.</a> Phytochemistry. 67 (21), 2341-2348 (2006).</li><li>Dani, V., Simon, W. J., Duranti, M., Croy, R. R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+tobacco+leaf+apoplast+proteome+in+response+to+salt+stress.">Changes in the tobacco leaf apoplast proteome in response to salt stress.</a> Proteomics. 5 (3), 737-745 (2005).</li><li>Delaunois, B., 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=Uncovering+plant-pathogen+crosstalk+through+apoplastic+proteomic+studies.">Uncovering plant-pathogen crosstalk through apoplastic proteomic studies.</a> Front Plant Sci. 5, 249 (2014).</li><li>Andreasson, E., Abreha, K. B., Resj&#246;, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+apoplast.">Isolation of apoplast.</a> Methods Mol. Biol. 1511, 233-240 (2017).</li><li>Lohaus, G., Pennewiss, K., Sattelmacher, B., Hussmann, M., Hermann, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+the+infiltration-centrifugation+technique+appropriate+for+the+isolation+of+apoplastic+fluid?+A+critical+evaluation+with+different+plant+species.">Is the infiltration-centrifugation technique appropriate for the isolation of apoplastic fluid? A critical evaluation with different plant species.</a> Physiol Plant. 111 (4), 457-465 (2001).</li><li>Nouchi, I., 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=Overcoming+the+difficulties+in+collecting+apoplastic+fluid+from+rice+leaves+by+the+infiltration-centrifugation+method.">Overcoming the difficulties in collecting apoplastic fluid from rice leaves by the infiltration-centrifugation method.</a> Plant Cell Physiol. 53 (9), 1659-1668 (2012).</li><li>Rumyantseva, N. I., Valieva, A. I., Kostyukova, Y. A., Ageeva, M. 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 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。

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  

760 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

A Method For Production of Recombinant mCD1d Protein in Insect Cells.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated heavy chains that are noncovalently associated with beta2-microglobulin. They bind lipids rather than peptides. Although their structure confirms the similarity of CD1 proteins to MHC class I and class II antigen presenting molecules, the mCD1d groove is relatively narrow, deep, and highly hydrophobic and it has two binding pockets instead of the several shallow pockets described for the classical MHC-encoded antigen-presenting molecules. Based upon their amino acid sequences, such a hydrobphobic groove provides an ideal environment for the binding of lipid antigens. The Natural Killer T (NKT) cells use their TCR to recognize glycolipids bound to or presented by CD1d. T cells reactive to lipids presented by CD1 have been involved in the protection against autoimmune and infectious diseases and in tumor rejection. Thus, the ability to identify, purify , and track the response of CD1-reactive NKT cell is of great importance . The generation of tetramers of alpha Galactosyl ceramide (a-Galcer) with CD1d has significant insight into the biology of NKT cells. Tetramers constructed from other CD1 molecules have also been generated and these new reagents have greatly expanded the knowledge of the functions of lipid-reactive T cells, with potential use in monitoring the response to lipid-based vaccines and in the diagnosis of autoimmune diseases and other treatments.

A Method to prepare Insect cells and infect them with baculovirus for the the purpose of production of recombinant mCD1d proteinand generating mCD1d tetramers.

重组昆虫细胞中的mCD1d的蛋白质的生产方法。

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated ...

科研

生物学

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

一个基于PCR的基因分型方法来区分野生型和观赏品种白茅</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

一个协议，用于基于蛋白质 - 蛋白质相互作用的鉴定<sup&gt; 15</sup&gt; N代谢标记，免疫沉淀法，的定量质谱法和亲和调制

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

植物蛋白复合体的翻译后修饰的鉴定

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

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. ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

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 ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

拟南芥种子高通量灭菌的标准化方法

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

Rice stripe virus (RSV), which causes significant economic loss of agriculture in East Asia, entirely depends on insect vectors for its effective transmission among host rice. Laodelphax striatellus (small brown planthopper, SBPH) is the primary insect vector that horizontally transmits RSV while sucking sap from the phloem. Saliva plays a significant role in insects' feeding behavior. A convenient method that will be useful for research on insects' saliva with piercing-sucking feeding behavior is described here. In this method, insects were allowed to feed on an artificial diet sandwiched between two stretched paraffin film layers. The diet containing the saliva was collected each day, filtered, and concentrated for further analysis. Finally, the quality of collected saliva was examined by protein staining and immunoblotting. This method was exemplified by detecting the presence of RSV and a mucin-like protein in the saliva of SBPH. These artificial feeding and saliva collection method will lay a foundation for further research on factors in insect saliva related to feeding behavior and virus transmission.

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

The present protocol describes a method to collect sufficient saliva from piercing-sucking insects using an artificial medium. This is a convenient method for collecting insect saliva and studying salivary function on insect feeding behavior and vector-borne virus transmission.