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

Biz teknik yardım için Lynn forlow-JECH ve Dannialle LeRoy teşekkür ederim. Bu makale amacıyla sadece bir ticaret adları veya ticari ürünlerin JAF ve JJH Mansiyon için Bitki Koruma ve Karantina [Proje # 2020-22620-022-00D] - Bu çalışma USDA ARS taban -CRIS fon, Ulusal Program 304 tarafından desteklenmiştir spesifik bilgi sağlama ve ABD Tarım Bakanlığı tarafından tavsiye veya onaylandığı anlamına gelmez. USDA bir fırsat eşitliği sağlayıcı ve işverendir.

Ifade plazmitlerinin yapımı 1. OE-PCR, Not: OE-PCR kullanılan tüm primerler için Tablo 1&#39;e bakınız. yüksek doğrulukta bir DNA polimerazı kullanımı bütün amplifikasyonlar için tavsiye edilir. Bununla birlikte, bu enzimler, genellikle bırakmak çünkü bir 3&#39; A, bir Taq DNA polimeraz ile kısa olmayan amplifiye inkübasyon gerçekleştirmek için gerekli olan &#39;A-kuyruk&#39; PCR ürünleri bir TA böcek hücresi ekspresyon içerisine klonlanması önce...

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Heterolog protein ekspresyon sistemleri çok sayıda alt uygulamalarda 4&#39;te kullanılan rekombinant proteinlerin üretimi için önemli bir araçtır. Mevcut çeşitli ifade sistemlerinden seçme ilgi protein için nihai hedef bağlıdır. Çeşitli böcek hücresi ifade sistemleri prokaryotik ve ökaryotik hücre ekspresyon sistemleri 5, 6 esnek alternatifler sunan mevcuttur. Protein ifadesini sürmek için bakulovirüs enfeksiyonu g...

böcek hücresi ifade sistemleri kullanılarak, rekombinant proteinlerin üretimi ökaryotik proteinlerin çalışma için sayısız avantajlar sunmaktadır. Yani, böcek hücreleri de benzer post-translasyonel modifikasyonları, işleme ve uygun biçimde katlanmış proteinler, 1, 2, 3 üretilmesi için avantajlı olan memeli hücrelerinde mevcut olanlar gibi ayırma mekanizmalara sahiptirler. Böcek hücre sistemleri, aynı zamanda, tipik olarak memeli hücre çizgileri 4, 5 den bakım için daha az kaynak ve daha az zaman ve çaba gerektirir. bakulovirüs ekspresyon sistemi, protein karakterizasyonu ve terapötik, yabancı peptidler ve aşı üretimi için viral proteinlerin imünojenik sunumu, çok sentezi için rekombinant proteinlerin üretimi de dahil olmak üzere artık birçok disiplinlerde kullanılan bir böcek hücre bazlı bir sistem, bir proteini kompleksleriVb glikosile proteinlerin üretimi. 1, 2, 4, 6. Bununla birlikte, çeşitli durum da baculovirüs ifade 3, 7 uygulanabilir olmayabilir ve nonlytic ve geçici böcek ifade sistemlerinin kullanılması daha uygun olabilir. Özellikle, geçici böcek hücresi ekspresyon rekombinant proteinin hızlı sentezi için imkanı sunar, viral-uygulanan hücre lizizi içermeyen az gelişim ve bakım gerektirir ve daha iyi bir protein sentezi 7, 8, 9 boyunca hücre hareketini incelemek için bir araç sağlar, 10. Bu protokol kullanılarak ekspresyon vektörlerinin hızla oluşturulmasını tarif etmektedir, iki aşamalı üst üste gelen uzatma PCR ile (OE-PCR) Escherichia coli içinde plazmid DNA standart klonlama. Plazmidler, çift transfekte ticari olarak temin edilebilen Kültürlenmiş böcek hücreleri kullanılır ve temsili proteinleri üretmek için. Protokol, iki farklı floresan etiketli hücre içi işaretleme proteinleri üretim ve kullanımını tarif eder ve böcek Bemisia tabaci iki aquaporin proteinleri ile ko göstermektedir. Aşağıdaki protokol, OE-PCR, hedef proteinlerin hücresel lokalizasyon amacıyla böcek hücre bakım ve transfeksiyon, ve floresan mikroskobu için temel bir yöntem sağlar.

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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

Heterolog protein ekspresyon sistemler, rekombinant proteinlerin üretimi için kullanılan, selüler trafik / lokalizasyonu yorumlanması ve alt organizma seviyesinde protein biyokimyasal fonksiyonunun belirlenmesi. bakulovirüs ifade sistemleri giderek sayısız biyoteknolojik, ilaç ve endüstriyel uygulamalar, var açık yararlar viral enfeksiyon içermeyen ama çoğu zaman göz ardı ve kullanılamayan ama nonlytic sistemlerde protein üretimi için kullanılmasına rağmen. Burada, nonlytic ekspresyon vektörleri ve geçici rekombinant protein ekspresyonunu oluşturmak için bir yöntem tarif eder. Bu protokol, rekombinant proteinlerin etkin hücresel lokalizasyonu için izin verir ve hızlı bir şekilde hücre içinde protein trafiğini ayırt etmek için kullanılabilir. Biz de, böcek Bemisia tabaci iki aquaporin proteinler de dahil olmak üzere, ticari olarak mevcut böcek hücresi hattı dört rekombinant proteinlerin ekspresyonu gösterirHücre plazma zarının spesifik hücre içi markör proteinler olarak ve hücre içi lizozomlar için. Yeniden birleştirici tüm proteinler rekombinant proteinlerin doğrudan teşhis edilmesine izin verir, karboksil terminallerinde, floresan protein işaretleri ile kimeraları üretildi. plazmidler ilgi ve bilinen bir alt hücresel markör genler için konstruktlarını barındıran hücrelerin çift transfeksiyon canlı hücre görüntüleme ve hücresel protein lokalizasyonu geliştirilmiş doğrulama sağlar.

OE-PCR, OE-PCR, bir kez çıkar ve floresan markör proteinin herhangi bir test genine karşılık gelen rekombinant kimerik proteinlerin üretimi için izin bir ekspresyon vektörüne yerleştirilir, kimerik DNA ürünleri sentezine izin verir. Şekil 1, floresan protein markör EGFP ile çerçeve dizileri (BtDrip1 ve BtDrip2_v1) kodlama B. tabaci aquaporin içeren pIB ifade vektörlerinin üretimi içi...

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 böcek hücresi ifade sistemleri üretim, selüler trafik / lokalizasyonu ve rekombinant protein, fonksiyonel analiz için atıl edilir. Burada, ticari olarak temin edilebilen bir lepidopteran hücresi çizgilerinde ifade vektörleri ve daha sonra geçici protein sentezlenmesinin üretilmesi için yöntemleri tarif eder. Hücre içi fluoresan işaretleyici proteinleri ile Bemisia tabaci aquaporins eş yerleşimi de sunulmuştur.

Kültürlü Böcek Hücrelerinde Geçici İfade ve Rekombinant Proteinlerin Hücresel Yerelleştirme

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.

Rekombinant mCD1d Böcek Hücrelerde Protein Üretimi İçin Bir Yöntem.

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

Organik Solvent ve Asetik Asit olmadan Jeller Coomassie G-250 ile Proteinlerin Boyama

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

Rekombinant Proteinlerin Metilotropik maya İfade 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

Rekombinant Proteinlerin Yüksek-düzey Geçici İfade için Bitkiler Verimli Agroinfiltration

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

Verimli Üretimi ve Saflaştırılması Rekombinant murin kindlin-3 Biophysical Çalışmaları Böcek hücrelerinden

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

Üretilen Rekombinant Disülfid zengin Venom Proteinler Uygulanan Yüksek Randımanlı Sayısal Anlatım Tarama ve Arıtma<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

Farelerde Hidrodinamik Gen Taşıyıcı Proteinlerin Geçici İfadesi

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

Kültür Memeli Hücrelerde Biyolojik tanımında ubikitin ve Ubiquitin benzeri Sistemi Regülatörleri tanımak siRNA Eleme

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

Bakteriler, Böcek Hücreleri ve Bitki Sistemleri: Rekombinant Protein Farklı Biofactories İfade Karşılaştırmalı Analizi

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the ...