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

Wij danken Lynn Forlow-Jech en Dannialle LeRoy voor technische ondersteuning. Dit werk werd ondersteund door base CRIS financiering van USDA ARS, Nationaal Programma 304 - Crop Protection and Quarantine [Project # 2020-22620-022-00D] om JAF en JJH Vermelding van handelsnamen of commerciële producten die in dit artikel is alleen voor het doel van het verstrekken van specifieke informatie en impliceert geen aanbeveling of goedkeuring door het Amerikaanse ministerie van Landbouw. USDA is een gelijke kans provider en werkgever.

1. OE-PCR voor de bouw van expressieplasmiden Opmerking: zie tabel 1 voor alle primers die in OE-PCR. Het gebruik van een high-fidelity DNA polymerase wordt aanbevolen voor alle amplificaties. Omdat deze enzymen vaak geen reactie 3&#39;A, is het noodzakelijk om een ​​korte, niet-amplificeren incubatie voeren met een Taq DNA polymerase &#39;A-staart&#39; van de PCR-producten voordat ze kloneren in een TA insectencel expressie vector. Dit protocol wordt een methode voor het ...

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Heterologe eiwitexpressie zijn belangrijk voor de vervaardiging van recombinante eiwitten gebruikt in talrijke afgeleide toepassingen 4. Het kiezen van de diverse expressiesystemen beschikbaar afhankelijk van het einddoel voor het eiwit van belang. Verschillende expressie in insectencellen systemen zijn beschikbaar die flexibele alternatieven voor prokaryote en eukaryote celexpressiesystemen 5, 6 te bieden. Insecten systemen die baculovir...

De productie van recombinante eiwitten met behulp van expressie in insectencellen systemen biedt tal van voordelen voor de studie van eukaryotische eiwitten. Namelijk insectencellen bezitten soortgelijke posttranslationele modificaties, verwerking en sorteermechanismen als die welke in zoogdiercellen, hetgeen voordelig is voor het produceren van correct gevouwen eiwitten 1, 2, 3. Insectencel systemen ook typisch minder middelen en minder tijd en moeite voor het onderhoud dan zoogdiercellijnen 4, 5. Het baculovirus expressiesysteem is zo&#39;n insectencellen gebaseerde systeem dat nu algemeen wordt gebruikt in vele disciplines, waaronder de productie van recombinante eiwitten voor eiwitkarakterisering en therapie, de immunogène presentatie van vreemde peptiden en virale eiwitten voor vaccinproductie, de synthese van multi -proteïne complexenDe productie van geglycosyleerde eiwitten, enz. 1, 2, 4, 6. Er zijn echter situaties waarin baculovirus expressie niet van toepassing 3, 7, en het gebruik van nonlytic en insecten transiënte expressiesystemen geschikter zijn. Bijzonder transiënte expressie in insectencellen biedt de mogelijkheid voor de snelle synthese van recombinant eiwit, vereist minder ontwikkeling en onderhoud, niet gepaard gaat met virale opgelegde cellysis en verschaft een middel om cellulaire transport beter bestuderen tijdens eiwitsynthese 7, 8, 9, 10. Dit protocol beschrijft de snelle vorming van expressievectoren gebruikt tweestaps overlap extension PCR (OE-PCR) Escherichia coli. Plasmiden worden gebruikt om dubbel transfecteren handel verkrijgbaar gekweekte insectencellen en representatieve eiwitten. Het protocol beschrijft de productie en het gebruik van twee verschillende fluorescent gelabelde subcellulaire markereiwitten en toont colocalisatie twee aquaporine eiwitten van het insect Bemisia tabaci. Het volgende protocol geeft de basismethode voor OE-PCR, insectencel onderhoud en transfectie en fluorescentiemicroscopie voor de cellulaire lokalisatie van doeleiwitten.

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

Heteroloog eiwit expressiesystemen worden gebruikt voor de productie van recombinante eiwitten, de interpretatie van cellulaire transport / lokalisatie en het vaststellen van de biochemische functie van eiwitten op sub-organismal niveau. Hoewel baculovirus expressiesystemen in toenemende mate gebruikt voor eiwitproductie in talrijke biotechnologische, farmaceutische en industriële toepassingen, nonlytic systemen die geen virale infectie inhouden duidelijke voordelen, maar vaak vergeten en onderbenut. Hier beschrijven we een werkwijze voor het nonlytic expressievectoren en transiënte expressie van recombinant eiwit. Dit protocol maakt een efficiënte cellulaire lokalisatie van recombinante eiwitten en kunnen worden gebruikt om snel te onderscheiden eiwittransport in de cel. We tonen de expressie van vier recombinante eiwitten in een in de handel verkrijgbaar insectencellijn, waaronder twee aquaporine eiwitten van insecten Bemisia tabaci, enals subcellulaire markereiwitten specifiek voor het cel plasmamembraan en intracellulaire lysosomen. Alle recombinante eiwitten werden geproduceerd als chimeren met fluorescerende eiwit markers aan hun carboxyl termini, die zorgt voor de directe detectie van de recombinante eiwitten. De dubbele transfectie van cellen met plasmiden constructen voor de genen van belang en een bekende subcellulaire marker zorgt voor live-cell imaging en verbeterde validatie van cellulaire eiwitten lokalisatie.

OE-PCR OE-PCR maakt de synthese van chimere DNA-producten die, wanneer in een expressievector ingebracht, maken de productie van recombinante chimere eiwitten overeenkomend naar proeven gen van belang en fluorescent merkereiwit. Figuur 1 een algemeen schema voor de bereiding van expressievectoren die pIB B. tabaci aquaporine coderende sequenties (BtDrip1 en BtDrip2_v1) in-frame met het fluorescerende pro...

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 insectencel expressiesystemen onderbenut productie, cellulair transport / lokalisatie en recombinante eiwitten functionele analyse. We beschrijven werkwijzen voor expressievectoren en transiënte daaropvolgende eiwitexpressie genereren handel verkrijgbaar Lepidoptera cellijnen. De co-localisatie van Bemisia tabaci aquaporins met fluorescente subcellulaire markereiwitten wordt gepresenteerd.

Transiënte expressie en cellulaire lokalisatie van recombinante eiwitten in gekweekte insectencellen

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.

Een methode voor de productie van recombinante eiwitten mCD1d in insectencellen.

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

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

Kleuring van eiwitten in Gels met Coomassie G-250 zonder organisch oplosmiddel en azijnzuur

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

Expressie van recombinante eiwitten in het methylotrofe gist Pichia pastoris</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

Efficiënte Agroinfiltration van planten voor High-level Tijdelijke expressie van recombinante eiwitten

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

Efficiënte productie en zuivering van recombinant muizen Kindlin-3 uit Insect Cellen voor Biofysische Studies

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

High Throughput Screening Kwantitatieve Expressie en zuivering Toegepast op Recombinant Disulfide-rijke Venom eiwitten die in<em&gt; E. coli</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

Voorbijgaande expressie van eiwitten door Hydrodynamic Gene Levering in Muizen

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 Screening naar Ubiquitin en Ubiquitin-achtig systeem regulatoren van biologische pathways identificeren in gekweekte zoogdiercellen

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

Een vergelijkende analyse van recombinant eiwit expressie in verschillende biofactories: Bacteriën, 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 ...