我们感谢林恩Forlow-Jech和丹尼尔尔·勒罗伊技术援助。植物保护检疫[项目＃2020-22620-022-00D]以商品名称或商品的JAF和JJH提及这篇文章仅仅是为目的 - 这项工作是由基地CRIS资金USDA ARS，国家304计划支持的提供具体资料，也没有通过美国农业部门暗示推荐或认可。美国农业部是一个平等的机会提供者和雇主。

1. OE-PCR用于表达质粒的构建注:请参阅表1在OE-PCR中使用的所有引物。建议对所有扩增使用高保真DNA聚合酶。然而，因为这些酶通常不留下3&#39; A，有必要用Taq DNA聚合酶进行简短的，非放大孵育至‘A-尾’的PCR产物将它们克隆到TA昆虫细胞表达前向量。此协议演示了一个方法，以产生昆虫表达质粒携带嵌合蛋白，与框融合到感兴趣的基因的羧基末端的荧光蛋白（在这种情况下，两个烟粉虱水通道蛋白的蛋白质），或亚细胞标记蛋白（ 图1）。 <ol><li> 使用OE-PCR，它由两个独立的轮扩增的，以产生:（1）编码感兴趣的基因的序列（ 烟粉虱水通道蛋白1:BtDrip1;框融合与称为EGFP或（2）的亚细胞标记物（ 果蝇性肽受体绿色荧光蛋白变体BtDrip2_v1）: 烟粉虱水通道蛋白2 DmSPR; 智人磷脂酶A2:HsPLA2）与所述的mCherry编码序列。直接结扎最终OE-PCR产品进入PIB / V5-他-TA质粒。 <ol><li>对于第一轮OE-PCR的，使用基因特异性有义引物（ 表1中以粗体示出）和嵌合反义引物（由AD和A&#39;-D"在图1中所示）（在表1斜体）对应利息/亚细胞标记物的基因的最后15个碱基（无终止密码子）和至所需的荧光蛋白的5&#39;端的15bp的（EGFP或mCherry的），以产生具有3&#39; 突出端的产物。 注:请参见表2 PCR条件。 </li><li>在一个单独的管中，使用基因特异性IC荧光蛋白反义引物包含来自感兴趣/亚细胞标记物的基因的3&#39;末端15个碱基和15个碱基对来自期望的荧光5&#39;端和嵌合有义引物（与表1中下划线示出）蛋白质以产生具有一个5&#39; 突出端的产物。 注:请参阅表2为PCR条件。将PCR模板可以是一个序列验证PCR产物，或更优选地，DNA质粒携带所需序列。这里所描述的研究使用了序列验证质粒。 </li><li>分离，用1％琼脂糖凝胶电泳PCR产物（使用标准的分子级琼脂糖）。 </li><li>消费和纯化预期大小（ 见表2），使用市售的DNA凝胶提取试剂盒的产品（见材料的表中这个协议所使用的特定试剂盒）。 </li><li>使用凝胶纯化的PCR产物从步骤1.1.4以产生最终的重叠Èxtension产品（ -在图1中&#39;A所示&#39;&#39; D&#39;）。使用基因特异性有义引物对利息/亚细胞标记物的基因和用于荧光蛋白以产生期望的嵌合序列对应的基因特异性反义引物。 注:请参阅表2为PCR条件。它可能是必要的经验确定的第一轮重叠延伸产物的最佳量添加到反应混合物中以产生最终的，全长嵌合PCR产物。 </li><li>分离由1％琼脂糖凝胶电泳第二轮OE-PCR产物。使用市售的DNA凝胶提取试剂盒消费和纯化产品。 </li><li>孵育凝胶纯化第二轮OE-PCR产物用1个单位Taq DNA聚合酶预混的10分钟在72℃，以产生3&#39;腺苷酸化（ 即 A尾）所需的TA克隆产品。 </li><li>结扎造成的腺苷酸化产品到PIB表达载体并使用标准的分子克隆技术来转化化学感受（热休克）或电感受态（电穿孔） 大肠杆菌细胞中。在含有合适的选择标记（ 即，氨苄青霉素或羧苄青霉素）的培养基平板过夜。 </li><li>使用载体特异性引物和特定的插入引物，以确定已用表达质粒窝藏在5&#39;-3&#39;方向插入插入阳性集落进行菌落PCR 12。准备克隆产生预期大小的PCR产物的过夜培养。 </li><li>隔离使用根据制造商的说明可商购的DNA纯化试剂盒（参见材料 表对该协议所使用的特定试剂盒）质粒DNA。验证由直接DNA测序每个插入的序列完整性。 </li></ol></li></ol>注:为了保持无菌条件下，进行要求的组织培养烧瓶中的层流罩内的开口的所有细胞的操作。打开层流罩紫外线杀菌灯至少1个小时的细胞的操作之前。穿丁腈手套及其使用之前净化替补，移液管，器皿，管，并用70％乙醇的烧瓶的表面上。熟悉基本的细胞培养技术，推荐13。 <ol><li>使用Tni的细胞（来自粉纹夜蛾的卵巢组织来源的建立的细胞培养物线），它们适应于无血清培养基中并予以保持在无血清昆虫培养基在28℃下在T25组织培养瓶中贴壁单层培养。 </li><li>保持的Sf9细胞（来自草地夜蛾卵巢组织来源的建立的细胞培养系）类似，但使用TNM-FH昆虫培养基补充编有10％胎牛血清（FBS）。 </li><li>通过从-80℃取出的库存小瓶中并允许它们在37℃水浴中解冻种子从冷冻的Sf9或Tni的细胞的原始培养物。消毒用70％乙醇的小瓶解冻后，将它们放在冰上。 </li><li>添加4mL的昆虫细胞培养基到一个新的T25烧瓶，并转移1mL的解冻的昆虫细胞悬浮液。将烧瓶置于28℃，非湿润的培养箱和允许细胞30-45分钟附着。 </li><li>替换用5mL合适的培养基的接种培养基和培养瓶转移到28℃的非加湿培养箱。每天监测细胞汇合。通道的细胞，当他们达到90％汇合。 注意:T25烧瓶的完全覆盖对应于〜5×10个6个细胞。 </li><li> 昆虫细胞传代。 <ol><li>到通道中的细胞，首先除去从含有使用无菌5mL的血清吸液管汇合的细胞将烧瓶中的耗尽介质。使得介质流向一个角，从细胞单层远离倾斜烧瓶中。小心地取出使用移液管的介质，而不干扰细胞。 </li><li>轻轻漂洗使用新的，无菌，5mL的血清吸液管包含用4mL无血清昆虫培养基的汇合的单层的T25烧瓶逐出Tni的细胞。移动跨越烧瓶中的移液管尖端，慢慢灌溉以除去松散附着在烧瓶底部的细胞。 <ol><li>通过删除所有媒体，翻转烧瓶中，并观察该瓶的底部是明确检查细胞充分分离。 </li></ol></li><li>为Sf9细胞，其附着越紧，加入4毫升新鲜TNM-FH培养基中，并用细胞刮棒以移去贴壁细胞。使用5毫升血清吸管轻轻混匀并减少细胞聚集。 </li><li>使用自动细胞计数器来估计每介质的体积活昆虫细胞的数目。转移0.1mL的细胞/培养基混合物的至1​​.5mL的微量离心管中。在一个单独的0.5毫升微量离心管中，细胞/培养基混合物的添加10μL至台盼蓝的10μL。 </li><li>从包装中取出细胞计数器室滑动件和添加细胞/培养基/台盼蓝混合液10μL于计数滑动的每一侧上。插入幻灯片进入细胞计数器和确定细胞密度和生存力。 </li><li>使用的细胞密度，计算并添加昆虫细胞培养基（最多5 mL）添加到新的无菌T25烧瓶的适当体积。 </li><li>输送约1-1.5×10 6个细胞用新鲜培养基T25烧瓶;标记烧瓶与细胞系，日期，介质使用时，加入的细胞数，和通道号（P N + 1的生成，其中，P n是上一代细胞的传代次数）;并将其放置在一个28℃的培养箱中培养瓶长达72小时。 注意:昆虫细胞可以连续地传播，尽管细胞可以是转染和/或异源更少接受后30代蛋白质表达。治疗与10％漂白溶液中的所有丢弃细胞/培养基和处置前高压灭菌的一次性塑料器皿。 </li></ol></li></ol> 3.昆虫细胞转染<ol><li>播种T25烧瓶高达1×10 6个或Tni的Sf9细胞在合适的昆虫细胞培养基（对于TN1和TNM-FH为的Sf9无血清昆虫培养基）并生长至融合72小时在28℃。 </li><li>取下并丢弃旧的介质，并用4毫升新鲜的无血清昆虫培养基移出细胞（参见步骤2.6.2和2.6.3，上文）。 </li><li>估计使用自动细胞计数器（参照步骤2.6.4，上文）的细胞密度。 </li><li>添加约7×10 5细胞至个体35毫米玻璃底培养皿和允许细胞在28℃下20-25分钟附着。 </li><li>对于每次转染，加入2微克质粒DNA（从一个质粒用于转染单或2微克来自各两个质粒用于做的在无菌的1.5mL微量离心管uble转染）到0.1毫升无血清昆虫培养基（无FBS两者的Sf9和Tni的转染）的。 </li><li>在一个单独的管中，混合8μL转染试剂用0.1mL无血清昆虫培养基中，然后该溶液转移到含有目的质粒DNA的管中。轻轻涡，并在室温孵育20 - 30分钟。 </li><li>稀释从步骤3.5将质粒转染混合物和3.6用0.8毫升无血清昆虫培养基，使得总体积等于1毫升。 </li><li>小心地从含有贴壁细胞的玻璃培养皿取出介质。覆盖贴壁细胞与稀释的质粒的转染培养基。 </li><li>孵育细胞在28℃5小时。 注意:用于转染的条件要求实现最大转染效率（经验优化例如，转染过夜而不是5小时，质粒DNA的使用量，将转染试剂的化学性质使用的， 等 ）。 </li><li>卸下并丢弃转染培养基，轻轻地用1mL无血清昆虫培养基洗细胞，小心不要以移去细胞。 </li><li>加入2毫升的新鲜昆虫细胞培养基（对于TN1和TNM-FH为的Sf9无血清昆虫培养基）中，并在28℃下孵育48-72小时。 注:同样，条件需要最大的异源蛋白表达经验优化。 </li></ol> 4.共聚焦荧光显微镜<ol><li>在48-72小时后转染，用1mL IPL-41昆虫培养基洗细胞一次，然后用2mL的IPL-41用于成像覆盖。 注意:此洗涤步骤减少了与在正常细胞的维持使用昆虫细胞介质中观察到的背景自发荧光。 </li><li>加入4滴Hoechst的活细胞染色试剂（见的用于本协议中使用的特定的核染色材料的表）的培养基中并在28℃下孵育20-25分钟。 注:其他NUC利尔污渍可以被取代，尽管染料应具有EGFP和的mCherry独特荧光分布。 </li><li>放置35mm培养皿到自封闭的激光扫描共聚焦显微镜（见材料的表中这个协议所使用的特定仪器）。 </li><li>调整的Hoechst，EGFP，和mCherry的观察条件的显微镜:Hoechst的激发/发射 - 四百六十一分之三百五十九纳米; EGFP激发/发射 - 510分之489纳米; mCherry的激发/发射 - 六百十分之五百八十〇纳米。 </li><li>使用10x物镜，以确认荧光表达，然后使用60X相衬水浸物镜切换为扫描模式下执行的初始扫描。 </li><li>调整激光功率（5-7％），检测器灵敏度（47-49％），扫描速度，Z轴的深度，和数码变焦，以优化图像对比度和分辨率。图像将细胞在1.5X数码变焦，得到总90X放大。 注:显微镜参数需要优化经验调整图像采集，并且可以具体到使用仪器。 </li><li>导出原始数据为TIFF图像文件，并修改（作物和覆盖）的数字产生。 </li></ol>

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Biol. 45, 125-140 (2014).</li><li>Lu, M., 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=A+baculovirus+(Bombyx+mori+nuclear+polyhedrosis+virus)+repeat+element+functions+as+a+powerful+constitutive+enhancer+in+transfected+insect+cells.">A baculovirus (Bombyx mori nuclear polyhedrosis virus) repeat element functions as a powerful constitutive enhancer in transfected insect cells.</a> J. Biol. Chem. 272, 30724-30728 (1997).</li><li>Ren, L., 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=Comparative+analysis+of+the+activity+of+two+promoters+in+insect+cells.">Comparative analysis of the activity of two promoters in insect cells.</a> African J. Biotechnol. 10, 8930-8941 (2011).</li><li>Snapp, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+proteins:+a+cell+biologist's+user+guide.">Fluorescent proteins: a cell biologist's user guide.</a> Trends Cell Biol. 19, 649-655 (2009).</li><li>Kohnhorst, C. L., 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=Subcellular+functions+of+proteins+under+fluorescence+single-cell+microscopy.">Subcellular functions of proteins under fluorescence single-cell microscopy.</a> Biochim. Biophys. Acta. 1864, 77-84 (2016).</li><li>Zinchuk, V., Grossenbacher-Zinchuk, O. <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+quantitative+colocalization+analysis:+focus+on+neuroscience.">Recent advances in quantitative colocalization analysis: focus on neuroscience.</a> Prog. Histochem. Cytochem. 44, 125-172 (2009).</li><li>Shaner, N. 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=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nat. Methods. 2 (12), 905-909 (2005).</li><li>Chalfie, M., 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+fluorescent+protein+as+a+marker+for+gene+expression.">Green fluorescent protein as a marker for gene expression.</a> Science. 263 (5148), 802-805 (1994).</li><li>Shaner, N. 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=Improved+monomeric+red,+orange+and+yellow+fluorescent+proteins+derived+from+Discosoma+sp.+red+fluorescent+protein.">Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.</a> Nat. Biotech. 22 (12), 1567-1572 (2004).</li></ol>

作者宣称，他们没有竞争的经济利益。

异源蛋白质表达系统可用于产生在许多下游应用4中使用的重组蛋白的重要工具。从现有的各种表达系统选择取决于感兴趣的蛋白质的最终目标。几个昆虫细胞表达系统是可用的提供灵活的替代原核和真核细胞表达系统5,6。需要杆状病毒感染，以驱动蛋白表达的昆虫系统是目前最流行的和广泛使用的昆虫系统中，用在研究和治疗应用<sup...

生产用昆虫细胞表达系统重组蛋白的真核蛋白质的研究提供了许多好处。即，昆虫细胞具有相似的翻译后修饰，处理和排序机制的那些存在于哺乳动物细胞中，这是有利的用于制备正确折叠的蛋白1，2，3。昆虫细胞系统通常还需要更少的资源和较少的时间和精力用于维护比哺乳动物细胞系4,5。杆状病毒表达系统是一种这样的昆虫细胞为基础的系统，它现已被广泛地用于许多学科，包括生产用于蛋白质表征和治疗剂，外源肽和用于疫苗生产的病毒蛋白的免疫原性的介绍，多合成重组蛋白的 - 蛋白质复合物，生产糖基化蛋白等 。 1，2，4，6。有，但是，情况，其中杆状病毒表达可能不适用3,7，以及使用nonlytic和瞬态昆虫表达系统的可能更合适。具体而言，瞬态昆虫细胞表达提供了重组蛋白的快速合成的可能性，需要更少的开发和维护，不涉及病毒强加细胞裂解，并提供了蛋白质合成7，8，9中更好地研究细胞运输的手段， 10。 这个协议描述了使用快速生成的表达载体的两步骤的重叠延伸PCR（OE-PCR） <suP类="外部参照"&gt; 11和质粒DNA在大肠杆菌中标准的克隆。质粒被用于双重转染市售培养的昆虫细胞，并产生代表的蛋白质。该协议描述了两种不同的荧光标记的亚细胞标志物蛋白的生产和使用，并演示共定位与来自昆虫烟粉虱 2水通道蛋白的蛋白质。以下方案提供了用于OE-PCR，昆虫细胞维持和转染，而对于目标蛋白的细胞定位的荧光显微镜的基本方法。

<table><tbody><tr><td>KOD DNA Polymerase</td><td>EMD Millipore</td><td>71085-3</td><td>High-fidelity DNA polymerase used for PCR amplification of overlap extension PCR products</td></tr><tr><td>ExTaq DNA Polymerase</td><td>TaKaRa-Clontech</td><td>RR001B</td><td>DNA polymerase used for A-tailing of PCR products</td></tr><tr><td>EconoTaq PLUS GREEN 2x DNA Polymerase Master Mix</td><td>Lucigen</td><td>30033-1</td><td>DNA polymerase used for bacterial colony PCR</td></tr><tr><td>Biometra TProfessional Gradient Thermocycler</td><td>Biometra/LABRepCo</td><td>070-851</td><td></td></tr><tr><td>Agarose LE</td><td>Benchmark Scientific</td><td>A1705</td><td></td></tr><tr><td>SYBR Safe DNA Gel Stain</td><td>ThermoFisher</td><td>S33102</td><td></td></tr><tr><td>Montage DNA Gel Extraction Kit</td><td>EMD Millipore</td><td>LSKGEL050</td><td></td></tr><tr><td>pIB/V5-His TOPO TA Expression Kit</td><td>ThermoFisher</td><td>K89020</td><td>Contains components needed to clone overlap extension PCR products, including linearized and topoisomerase I-activated pIB/V5-His-TOPO vector, One Shot TOP10 chemically competent E. coli, and salt solution.</td></tr><tr><td>QIAprep Spin MiniPrep Kit</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>QIAcube Robotic Workstation</td><td>Qiagen</td><td>9001292</td><td></td></tr><tr><td>Purifier Vertical Clean Bench</td><td>Labconco</td><td>3970401</td><td></td></tr><tr><td>Tni cultured insect cell Line</td><td>Allele Biotech</td><td>ABP-CEL-10005</td><td></td></tr><tr><td>Sf9 cultured insect cell Line</td><td>Allele Biotech</td><td>ABP-CEL-10002</td><td></td></tr><tr><td>Serum-Free Insect Culture Medium</td><td>Allele Biotech</td><td>ABP-MED-10002</td><td></td></tr><tr><td>TNM-FH Insect Culture Medium</td><td>Allele Biotech</td><td>ABP-MED-10001</td><td></td></tr><tr><td>IPL-41 Insect Medium</td><td>ThermoFisher</td><td>11405081</td><td></td></tr><tr><td>Cellfectin II Transfection Reagent</td><td>ThermoFisher</td><td>10362100</td><td></td></tr><tr><td>16 cm Disposable Cell Scrapers</td><td>Sarstedt</td><td>83.1832</td><td>Cell scrapers with two-position blade</td></tr><tr><td>25 cm&lt;sup&gt;2&lt;/sup&gt; (T25) Tissue Culture Flasks with Vent Filter Caps</td><td>Life Science Products</td><td>CT-229331</td><td></td></tr><tr><td>Transfer Pipets</td><td>Fisher</td><td>1371120</td><td></td></tr><tr><td>Sterile, 50 mL Bio-Reaction Tubes</td><td>Life Science Products</td><td>CT-229475</td><td></td></tr><tr><td>PipetteBoy</td><td>VWR</td><td>14222-180</td><td></td></tr><tr><td>5 mL Serological Pipettes</td><td>Sarstedt</td><td>86.1253.001</td><td></td></tr><tr><td>0.5 mL Flat-Cap PCR Tubes</td><td>Fisher</td><td>14230200</td><td></td></tr><tr><td>Polypropylene Biohazard Autoclave Bags</td><td>Fisher</td><td>01828C</td><td></td></tr><tr><td>35 mm #1.5 Glass Bottom Dishes</td><td>Matsunami Glass</td><td>D35-14-1.5-U</td><td>35 mm dish diameter, 14 mm glass diameter, 1.5 mm glass thickness, uncoated</td></tr><tr><td>Incubator, Model 1510E</td><td>VWR</td><td>35823-961</td><td></td></tr><tr><td>Countess II FL Cell Counter</td><td>ThermoFisher</td><td>AMQAF1000</td><td></td></tr><tr><td>Countess Cell Counting Chamber Slides with 0.4% Trypan Blue Reagent</td><td>ThermoFisher</td><td>C10228</td><td></td></tr><tr><td>Fluoview FV10i-LIV Laser Scanning Confocal Microscope</td><td>Olympus</td><td>FV10i-LIV</td><td></td></tr><tr><td>HsPLA2/pCS6 plasmid DNA</td><td>transOMIC Technologies</td><td>TCH1303</td><td></td></tr><tr><td>pmCherry Vector</td><td>Clontech</td><td>632522</td><td></td></tr><tr><td>NucBlue Live ReadyProbes Reagent (Hoechst 33342)</td><td>ThermoFisher</td><td>R37605</td><td></td></tr></tbody></table>

transient expression and cellular localization of recombinant proteins in cultured insect cells

异源蛋白质表达系统用于生产重组蛋白，细胞运输/定位的解释，和蛋白质在子有机体水平的生化功能的确定。虽然杆状病毒表达系统在众多的生物技术，制药和工业应用中，不涉及病毒感染有明显的好处，但往往被忽视和未充分利用nonlytic系统越来越多地用于生产蛋白质。在这里，我们描述了用于产生nonlytic表达载体和瞬时重组蛋白表达的方法。该协议允许对重组蛋白的高效细胞定位和可用于在细胞内迅速辨别蛋白质运输。我们示出了四个重组蛋白在市售的昆虫细胞系的表达，包括来自昆虫烟粉虱 2水通道蛋白的蛋白质，以及作为特异于细胞质膜亚细胞标志物蛋白和细胞内溶酶体。所有的重组蛋白被生产为具有在它们的羧基末端，其允许直接检测重组蛋白的荧光蛋白标记的嵌合体。细胞与质粒构建携带针对感兴趣和已知的亚细胞标记物的基因的转染双允许活细胞成像，改进的蜂窝蛋白质定位的验证。

OE-PCR OE-PCR允许的是，一旦被插入到表达载体中，允许产生对应于目标和荧光标记蛋白的任何测试基因的重组嵌合蛋白的嵌合DNA产物的合成。 图1表示用于生产含有烟粉虱水通道蛋白编码符合读框的序列（BtDrip1和BtDrip2_v1）与荧光蛋白质标记EGFP PIB的表达载体的一般方案。在这种情况下，制备含有EGFP在它们的羧...

Watch this Scientific Journal Video about Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells at JoVE.com

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

Nonlytic昆虫细胞表达系统没有得到充分利用进行生产，细胞运输/定位，和重组蛋白质功能分析。这里，我们描述的方法来产生在市售鳞翅目细胞系的表达载体和随后的瞬时表达。与亚细胞荧光标记蛋白烟粉虱水通道蛋白的共定位还提出。

瞬时表达和重组蛋白的细胞定位在培养的昆虫细胞

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

transient-expression-cellular-localization-recombinant-proteins

Research

JoVE Journal

Biology

10.7K 次观看.  Agricultural Research Service, U.S. Department of Agriculture.  Nonlytic昆虫细胞表达系统没有得到充分利用进行生产，细胞运输/定位，和重组蛋白质功能分析。这里，我们描述的方法来产生在市售鳞翅目细胞系的表达载体和随后的瞬时表达。与亚细胞荧光标记蛋白烟粉虱水通道蛋白的共定位还提出。 

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

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as&#160;fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems.

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.

重组纤维素酶Cel5A从表达<em&gt;里氏木霉</em&gt;在烟草

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture ...

科研

环境科学

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

通过诱导瞬时转染可控离子通道表达

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

遗传学

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell culture system. This system was developed to express foreign genes under the control of the baculoviral promoter in transient expression plasmids. Furthermore, this system can be applied to a functional assay of either the baculovirus itself or foreign proteins. The most widely and commercially available transient gene expression system is developed based on the immediate-early gene (IE) promoter of Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). However, a low expression level of foreign genes in insect cells was observed. Therefore, a transient gene expression system was constructed for improving protein expression. In this system, recombinant plasmids were constructed to contain the target sequence under the control of the Drosophila heat shock 70 (Dhsp70) promoter. This protocol presents the application of this heat shock-based pDHsp/V5-His (V5 epitope with 6 histidine)/Spodoptera frugiperda cell (sf9 cell) system; this system is available not only for gene expression but also for evaluating the anti-apoptotic activity of candidate proteins in insect cells. Furthermore, this system can be either transfected with one recombinant plasmid or co-transfected two potentially functionally antagonistic recombinant plasmids in insect cells. The protocol demonstrates the efficiency of this system and provides a practical case of this technique.

Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

This protocol describes a heat shock-induced protein expression system (pDHsp/V5-His/sf9 cell system), which can be used for either expressing foreign proteins or evaluating the anti-apoptotic activity of potential foreign proteins and their truncated amino acids in insect cells.

昆虫细胞 (sf9) 外源基因在蛋白质功能检测中的瞬态表达

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell ...

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The baculovirus-mediated insect cell expression system is one of the systems used to express recombinant eukaryotic secretory proteins with some post-translational modifications. The secretory proteins need to be routed through the secretory pathways for protein glycosylation, disulfide bonds formation, and other post-translational modifications. To improve the existing insect cell expression of secretory plant proteins, a baculovirus expression vector is modified by the addition of either a GP67 or a hemolin signal peptide sequence between the promoter and multiple-cloning sites. This newly designed modified vector system successfully produced a high yield of soluble recombinant secreted plant receptor proteins of Arabidopsis thaliana. Two of the expressed plant proteins, the extracellular domains of Arabidopsis TDR and PRK3 plasma membrane receptors, were crystallized for X-ray crystallographic studies. The modified vector system is an improved tool that can potentially be used for the expression of recombinant secretory proteins in the animal kingdom as well.

Here, we present the protocols for utilizing the insect cell and baculovirus protein expression system to produce large quantities of plant secreted proteins for protein crystallization. A baculovirus expression vector has been modified with either GP67 or insect hemolin signal peptide for plant protein secretion expression in insect cells.

改进杆状病毒表达载体生产昆虫细胞分泌植物蛋白

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The ...

生物化学

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

一个从人体细胞分泌蛋白生产的方便，一般的表达平台

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

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 ...

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

重组蛋白表达的结构生物学在HEK 293F悬浮细胞:一种新的和可访问方法

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

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 ...

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

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

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

瞬时表达和重组蛋白的细胞定位在培养的昆虫细胞

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