Name: transient expression and cellular localization of recombinant proteins in cultured insect cells
Uploaded: 2017-04-20T15:00:00
Description: Watch this Scientific Journal Video about Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells at JoVE.com

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...

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	<li>Kost, T. A., 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=Baculovirus+as+versatile+vectors+for+protein+expression+in+insect+and+mammalian+cells.">Baculovirus as versatile vectors for protein expression in insect and mammalian cells.</a> Nat. Biotechnol. 23 (5), 567-575 (2005).</li><li>van Oers, M. 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=Thirty+years+of+baculovirus-insect+cell+protein+expression:+from+dark+horse+to+mainstream+technology.">Thirty years of baculovirus-insect cell protein expression: from dark horse to mainstream technology.</a> J. Gen. Virol. 96 (1), 6-23 (2015).</li><li>Contreras-G&#242;mez, A., 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=Protein+production+using+the+baculovirus-insect+cell+expression+system.">Protein production using the baculovirus-insect cell expression system.</a> Biotechnol. Progr. 30 (1), 1-18 (2014).</li><li>Hunt, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+gene+to+protein:+a+review+of+new+and+enabling+technologies+for+multi-parallel+protein+expression.">From gene to protein: a review of new and enabling technologies for multi-parallel protein expression.</a> Protein Expres. Purif. 40 (1), 1-22 (2005).</li><li>Kollewe, C., Vilcinskas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+recombinant+proteins+in+insect+cells.">Production of recombinant proteins in insect cells.</a> Am. J. Biochem. Biotechnol. 9 (3), 255-271 (2013).</li><li>Altmann, F., Berger, E. G., Clausen, H., Cummings, R. D., 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=Insect+cells+as+hosts+for+the+expression+of+recombinant+glycoproteins.">Insect cells as hosts for the expression of recombinant glycoproteins.</a> Glycotechnology. , 29-43 (1999).</li><li>Shen, X., 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+simple+plasmid-based+transient+gene+expression+method+using+High+Five+cells.">A simple plasmid-based transient gene expression method using High Five cells.</a> J. Biotechnol. 216, 67-75 (2015).</li><li>Chen, 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=Rapid+screening+of+membrane+protein+expression+in+transiently+transfected+insect+cells.">Rapid screening of membrane protein expression in transiently transfected insect cells.</a> Protein Expres. Purif. 88 (1), 134-142 (2013).</li><li>Shen, X., 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=Virus-free+transient+protein+production+in+Sf9+cells.">Virus-free transient protein production in Sf9 cells.</a> J. Biotechnol. 171, 61-70 (2014).</li><li>Loomis, K. 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=InsectDirect+System:+rapid,+high-level+protein+expression+and+purification+from+insect+cells.">InsectDirect System: rapid, high-level protein expression and purification from insect cells.</a> J. Struct. Funct. Genomics. 6 (2), 189-194 (2005).</li><li>Wurch, 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=A+modified+overlap+extension+PCR+method+to+create+chimeric+genes+in+the+absence+of+restriction+enzymes.">A modified overlap extension PCR method to create chimeric genes in the absence of restriction enzymes.</a> Biotechnol. Tech. 12 (9), 653-657 (1998).</li><li>Woodman, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+PCR+of+intact+bacteria+(colony+PCR).">Direct PCR of intact bacteria (colony PCR).</a> Curr. Protoc. Microbiol. 9 (3), 1-6 (2008).</li><li>Mathew, L. 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=Identification+and+characterization+of+functional+aquaporin+water+channel+protein+from+alimentary+tract+of+whitefly,+Bemisia+tabaci.">Identification and characterization of functional aquaporin water channel protein from alimentary tract of whitefly, Bemisia tabaci.</a> Insect Biochem. Mol. Biol. 41 (3), 178-190 (2011).</li><li>Van Ekert, E., 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=Molecular+and+functional+characterization+of+Bemisia+tabaci+aquaporins+reveals+the+water+channel+diversity+of+hemipteran+insects.">Molecular and functional characterization of Bemisia tabaci aquaporins reveals the water channel diversity of hemipteran insects.</a> Insect Biochem. Mol. Biol. 77, 39-51 (2016).</li><li>Hull, J. J., Brent, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+sex+peptide+receptor-like+transcript+from+the+western+tarnished+plant+bug+Lygus+hesperus.">Identification and characterization of a sex peptide receptor-like transcript from the western tarnished plant bug Lygus hesperus.</a> Insect Mol. Biol. 23 (3), 301-319 (2014).</li><li>Maroniche, G. A., 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=Development+of+a+novel+set+of+Gateway-compatible+vectors+for+live+imaging+in+insect+cells.">Development of a novel set of Gateway-compatible vectors for live imaging in insect cells.</a> Insect Mol. Biol. 20 (5), 675-685 (2011).</li><li>Hull, J. 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=Identification+of+the+western+tarnished+plant+but+(Lygus+hesperus)+olfactory+co-receptor+ORCO:+Expression+profile+and+confirmation+of+atypical+membrane+topology.">Identification of the western tarnished plant but (Lygus hesperus) olfactory co-receptor ORCO: Expression profile and confirmation of atypical membrane topology.</a> Arch. Insect Biochem. 81 (4), 179-198 (2012).</li><li>Lee, J. 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=Re-evaluation+of+the+PBAN+receptor+molecule:+Characterization+of+PBANR+variants+expressed+in+the+pheromone+glands+of+moths.">Re-evaluation of the PBAN receptor molecule: Characterization of PBANR variants expressed in the pheromone glands of moths.</a> Front. Endocrinol. 3 (6), 1-12 (2012).</li><li>Fabrick, J. A., 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=Molecular+and+functional+characterization+of+multiple+aquaporin+water+channel+proteins+from+the+western+tarnished+plant+bug,+Lygus+hesperus.">Molecular and functional characterization of multiple aquaporin water channel proteins from the western tarnished plant bug, Lygus hesperus.</a> Insect Biochem. Mol. 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>

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。

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		<tr height="21">
			<td height="21" style="height:21px;width:425px;">KOD DNA Polymerase</td>
			<td style="width:164px;">EMD Millipore</td>
			<td style="width:119px;">71085-3</td>
			<td style="width:645px;">High-fidelity DNA polymerase used for PCR amplification of overlap extension PCR products</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">ExTaq DNA Polymerase</td>
			<td style="width:164px;">TaKaRa-Clontech</td>
			<td style="width:119px;">RR001B</td>
			<td>DNA polymerase used for A-tailing of PCR products</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">EconoTaq PLUS GREEN 2x DNA Polymerase Master Mix</td>
			<td style="width:164px;">Lucigen</td>
			<td style="width:119px;">30033-1</td>
			<td>DNA polymerase used for bacterial colony PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Biometra TProfessional Gradient Thermocycler</td>
			<td style="width:164px;">Biometra/LABRepCo</td>
			<td style="width:119px;">070-851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Agarose LE</td>
			<td style="width:164px;">Benchmark Scientific</td>
			<td style="width:119px;">A1705</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">SYBR Safe DNA Gel Stain</td>
			<td>ThermoFisher</td>
			<td style="width:119px;">S33102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Montage DNA Gel Extraction Kit</td>
			<td style="width:164px;">EMD Millipore</td>
			<td style="width:119px;">LSKGEL050</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:425px;">pIB/V5-His TOPO TA Expression Kit</td>
			<td>ThermoFisher</td>
			<td style="width:119px;">K89020</td>
			<td style="width:645px;">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 height="21">
			<td height="21" style="height:21px;width:425px;">QIAprep Spin MiniPrep Kit</td>
			<td>Qiagen</td>
			<td style="width:119px;">27104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">QIAcube Robotic Workstation</td>
			<td>Qiagen</td>
			<td style="width:119px;">9001292</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Purifier Vertical Clean Bench</td>
			<td>Labconco</td>
			<td>3970401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tni cultured insect cell Line</td>
			<td>Allele Biotech</td>
			<td>ABP-CEL-10005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sf9 cultured insect cell Line</td>
			<td>Allele Biotech</td>
			<td>ABP-CEL-10002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serum-Free Insect Culture Medium</td>
			<td>Allele Biotech</td>
			<td>ABP-MED-10002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TNM-FH Insect Culture Medium</td>
			<td>Allele Biotech</td>
			<td>ABP-MED-10001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IPL-41 Insect Medium</td>
			<td>ThermoFisher</td>
			<td>11405081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellfectin II Transfection Reagent</td>
			<td>ThermoFisher</td>
			<td>10362100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">16 cm Disposable Cell Scrapers</td>
			<td>Sarstedt</td>
			<td>83.1832</td>
			<td>Cell scrapers with two-position blade</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">25 cm2 (T25) Tissue Culture Flasks with Vent Filter Caps</td>
			<td>Life Science Products</td>
			<td>CT-229331</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transfer Pipets</td>
			<td>Fisher</td>
			<td>1371120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile, 50 mL Bio-Reaction Tubes</td>
			<td>Life Science Products</td>
			<td>CT-229475</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PipetteBoy</td>
			<td>VWR</td>
			<td>14222-180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5 mL Serological Pipettes</td>
			<td>Sarstedt</td>
			<td>86.1253.001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5 mL Flat-Cap PCR Tubes</td>
			<td>Fisher</td>
			<td>14230200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polypropylene Biohazard Autoclave Bags</td>
			<td>Fisher</td>
			<td>01828C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Incubator, Model 1510E</td>
			<td>VWR</td>
			<td>35823-961</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Countess II FL Cell Counter</td>
			<td>ThermoFisher</td>
			<td>AMQAF1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Countess Cell Counting Chamber Slides with 0.4% Trypan Blue Reagent</td>
			<td>ThermoFisher</td>
			<td>C10228</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluoview FV10i-LIV Laser Scanning Confocal Microscope</td>
			<td>Olympus</td>
			<td>FV10i-LIV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HsPLA2/pCS6 plasmid DNA</td>
			<td>transOMIC Technologies</td>
			<td>TCH1303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pmCherry Vector</td>
			<td>Clontech</td>
			<td>632522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">NucBlue Live ReadyProbes Reagent (Hoechst 33342)</td>
			<td style="width:164px;">ThermoFisher</td>
			<td style="width:119px;">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 ...

Research

JoVE Journal

Biology

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

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

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

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

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

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

在甲醇酵母表达的重组蛋白毕赤酵母</em

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

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

高级别瞬态表达重组蛋白的植物高效的农杆菌

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

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

高效的生产和纯化重组小鼠Kindlin-3从昆虫细胞生物物理研究

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

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

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

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

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

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

蛋白通过流体动力基因传递在小鼠体内瞬时表达

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

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

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 ...