Name: modifying baculovirus expression vectors to produce secreted plant proteins in insect cells
Uploaded: 2018-08-20T15:00:00
Description: Watch this Scientific Journal Video about Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells at JoVE.com

Dette arbejde blev støttet af de nye Fakultet start midler fra North Carolina State University for Guozhou Xu.

Bemærk: Bruges en insekt celle/baculovirus system med modificerede udtryk vektorer for sekretoriske plante protein udtryk og krystallisering.
1. ændring af en Baculovirus udtryk vektor med GP67 Signal peptid for plante Protein sekretion udtryk
<ol><li>Syntetisere en DNA-fragment, indeholdende en 5' BglII skæring site13, GP67 sekretion signal sekvensen, og en multi-kloning site med NotI, BamHI, EcoRI, StuI, SalI, SpeI, XbaI, PstI og XhoI (figur ...

<ol>
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Betragtning af mangfoldigheden i størrelse og stabilitet af tusindvis af proteiner til stede i de biologiske systemer, det er ofte empirisk til et forskningsprojekt laboratorium for at afgøre, hvilke heterolog ekspressionssystem har at blive valgt til udtryk af et specifikt protein. E. coli udtryk system er ofte det første valg for protein udtryk på grund af den korte livscyklus for bakterier, lave omkostninger dyrkningsmedier og relativt let at skalere op19. For at udtrykke stor euka...

Det er bydende nødvendigt for et forskningslaboratorium at være i stand til at producere store mængder af homogene rekombinante proteiner til biokemiske og biofysiske beskrivelser, især for X-ray krystallografiske undersøgelser. Der er mange veletablerede Heterolog ekspression systemer såsom Escherichia coli, gær, insekt celler, pattedyrceller, planteceller, etc. blandt dem, baculovirus-medieret insekt celle udtryk system er en af mest almindeligt anvendte teknikker til at producere store mængder af strukturelt foldede store rekombinant eukaryote proteiner for protein krystallisering1.
Udtrykket vektorer af baculovirus udtryk system er udviklet til at indeholde en stærk polyhedrin eller P10 initiativtageren til at producere et højt udbytte af rekombinant intracellulære proteiner2,3. For at gøre en rekombinante baculovirus, klonet gen af interesse i et insekt vektor indeholdende polyhedrin (polh) locus af Autographa californica multi-nucleopolyhedroviral genom. Den resulterende konstruktion er derefter sekventeret og dets korrekte åben læsning ramme (ORF) er verificeret. Den rigtige konstruktion føres derefter ind i cellen vært insekt gennem processen med Transfektion. Gen af interesse er indsat i det virale genom af homologe rekombination. Denne begivenhed resulterer i produktionen af af rekombinante viral genomet, som derefter replikater at producere rekombinant okulerede virus partikler1.
De insekt celler, der er mest almindeligt anvendt i udtrykket systemet er Sf9 og høj fem (Hi5) celler. Sf9 celler er en klonal isolat af Sf21, stammer fra de puppe æggestokkene celler af Spodoptera frugiperda, og Hi5 celler er en klonal isolatet hidrører fra forældrenes Trichoplusia ni æggestokkene celle linje TN-3684,5. Co transfections, virus forstærkning og plaque assays er gennemført på Sf9 celler, mens Hi5 celler er typisk valgt at producere større mængder af rekombinante proteiner6. Det er værd at bemærke, at Hi5 cellerne ikke er egnet til generation og forstærkning af virus descendenter på grund af deres tendens til at producere mutant vira. Traditionelt, anses et temperaturområde på 25-30 ° C for at være god til dyrkning af insekt celler. Det er blevet rapporteret, at 27-28 ° C er den optimale temperatur for de insekt celle-vækst og infektion7,-8.
Indførelsen af et stærkt signal sekvensen forud genet er nødvendig for den høje udtryk for de udskilles proteiner. Signal sekvensen vil effektivt guide den oversatte rekombinante proteiner i det endoplasmatiske reticulum for protein sekretion og posttranslationelle ændringer nødvendige for ordentlig foldning og stabilisering3. Signal peptid sekvenser, er som baculovirus indhylle protein GP64/67, honningbien melittin og andre, blevet valgt til at lette udtrykket af sekretoriske rekombinante proteiner i baculovirus-medieret udtryk systemer3. Indførelsen af signal peptid af GP67 har vist sig at forbedre udtryk udbyttet af en udskilles rekombinante protein, i forhold til ved hjælp af den iboende signal peptid target gen9. Hemolin er en hemolymph protein af den gigantiske silke møl Hyalophora cecropia, fremkaldt af bakterier infektion10. På grund af det relativt høje niveau af inducerede udtryk, kan signal peptid sekvens af genet bruges til at mægle udtrykket sekretion af af rekombinante proteiner i baculovirus-insekt celle system.
A. thaliana Tracheary Element differentiering hæmmende faktor Receptor (TDR) og Pollen Receptor Kinase 3 (PRK3) begge tilhører familien plante leucin-rig Repeat Receptor-lignende Kinase (LRR-RLK) af proteiner11,12 . For at studere struktur og funktion af denne familie af plante receptorproteiner, samt at lette det strukturelle og biokemiske karakterisering af andre vegetabilske udskilles proteiner, er baculovirus-insekt celle udtryk system blevet ændret til forbedre protein kvalitet og produktion afkast. De ekstracellulære domæner af både TDR og PRK3 har med held været udtrykt ved hjælp af to modificerede udtryk vektorer i baculovirus-insekt celle udtryk system. Begge de ekstracellulære domæner i TDR og PRK3 proteiner har været krystalliseret. Denne artikel rapporterer den proteinekspression og -oprensning af store mængder af rekombinant udskilles vegetabilske proteiner med to modificerede baculovirus udtryk vektorer ved at indarbejde enten en GP67 eller en hemolin signal sekvensen mellem arrangøren og flere kloning websteder.

<table>
	<tbody>
		<tr>
			<td>Incubator shaker</td>
			<td>VWR</td>
			<td>Model Excella E25</td>
			<td>27 oC</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>VWR</td>
			<td>Model 2005</td>
			<td>27 oC</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>BECKMAN</td>
			<td>Model J-6</td>
			<td>with a swing bucket rotor</td>
		</tr>
		<tr>
			<td>Herculase II Fusion DNA Polymerase</td>
			<td>Agilent</td>
			<td>600677-51</td>
			<td>For PCR amplification of DNA</td>
		</tr>
		<tr>
			<td>Thermal Cycler</td>
			<td>BIO-RAD</td>
			<td>Model C1000 Touch</td>
			<td>For PCR amplification of DNA</td>
		</tr>
		<tr>
			<td>Incubator shaker</td>
			<td>New Brunswick Scientific</td>
			<td>Model I 24</td>
			<td>For growing baceria culture</td>
		</tr>
		<tr>
			<td>Customer DNA synthesis</td>
			<td>GENSCRIPT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BamHI (HF)</td>
			<td>New England Biolabs</td>
			<td>R3136S</td>
			<td>Restriction Endonuclease</td>
		</tr>
		<tr>
			<td>BglII</td>
			<td>New England Biolabs</td>
			<td>R0144S</td>
			<td>Restriction Endonuclease</td>
		</tr>
		<tr>
			<td>NotI (HF)</td>
			<td>New England Biolabs</td>
			<td>R3189S</td>
			<td>Restriction Endonuclease</td>
		</tr>
		<tr>
			<td>XhoI</td>
			<td>New England Biolabs</td>
			<td>R0146S</td>
			<td>Restriction Endonuclease</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>New England Biolabs</td>
			<td>M0202T</td>
			<td>DNA ligation</td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td>DNA purification from Agorase gel</td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td>Plasmid DNA purification from bacteria culture</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Thermo Fisher Scientific</td>
			<td>15510-019</td>
			<td>For DNA gel electropherosis</td>
		</tr>
		<tr>
			<td>MAX Efficiency DH5&#945; competen cell</td>
			<td>Invitrogen</td>
			<td>18-258-012</td>
			<td>For transformation of DNA ligation mixture</td>
		</tr>
		<tr>
			<td>Lennox L LB Broth</td>
			<td>Research Product International Corp.</td>
			<td>L24066-5000.0</td>
			<td>For making bacteria culture</td>
		</tr>
		<tr>
			<td>Ampicillin sodium salt</td>
			<td>Thermo Fisher Scientific</td>
			<td>11593-019</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Kanamycin Sulfate</td>
			<td>Thermo Fisher Scientific</td>
			<td>15160-054</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Tetracycline</td>
			<td>Thermo Fisher Scientific</td>
			<td>64-75-5</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15710-064</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>MAX Efficiency DH10Bac competent cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>10361-012</td>
			<td>For making bacmid DNA</td>
		</tr>
		<tr>
			<td>S.O.C. Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>15544-034</td>
			<td>For DNA transformation</td>
		</tr>
		<tr>
			<td>CellFECTIN II Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>10362-101</td>
			<td>Insect cell transfection reagent</td>
		</tr>
		<tr>
			<td>Bac-to-Bac Expression System</td>
			<td>Thermo Fisher Scientific</td>
			<td>10359-016</td>
			<td>Baculovirus-insect cells expression kit</td>
		</tr>
		<tr>
			<td>Bluo-gal</td>
			<td>Thermo Fisher Scientific</td>
			<td>15519-028</td>
			<td>For isolation of recominant Bacmid DNA</td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Thermo Fisher Scientific</td>
			<td>15529-019</td>
			<td>For isolation of recominant Bacmid DNA</td>
		</tr>
		<tr>
			<td>pFastBac1</td>
			<td>Thermo Fisher Scientific</td>
			<td>10360014</td>
			<td>Baculorirus expression vector</td>
		</tr>
		<tr>
			<td>Sf9 cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>11496015</td>
			<td>Sf9 monolayer cells</td>
		</tr>
		<tr>
			<td>Hi5 cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>B85502</td>
			<td>High Five insect cells</td>
		</tr>
		<tr>
			<td>Grace&#8217;s insect medium, unsupplemented</td>
			<td>Thermo Fisher Scientific</td>
			<td>11595030</td>
			<td>Sf9 cell transfection minimum medium</td>
		</tr>
		<tr>
			<td>Grace&#8217;s insect medium, supplemented</td>
			<td>Thermo Fisher Scientific</td>
			<td>11605102</td>
			<td>Sf9 monolayer cell culture complete medium</td>
		</tr>
		<tr>
			<td>Sf-900 II SFM</td>
			<td>Thermo Fisher Scientific</td>
			<td>10902104</td>
			<td>Sf9 suspension cell culture medium without FBS</td>
		</tr>
		<tr>
			<td>Express Five SFM</td>
			<td>Thermo Fisher Scientific</td>
			<td>10486025</td>
			<td>Hi5 cell culture medium</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>100 ml (10,000 I.U./ml)</td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030081</td>
			<td>100 ml</td>
		</tr>
		<tr>
			<td>FBS Certified</td>
			<td>Thermo Fisher Scientific</td>
			<td>16000-044</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>08-772-1B</td>
			<td>Flat-bottom</td>
		</tr>
		<tr>
			<td>150 mm plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>353025</td>
			<td>100/case</td>
		</tr>
		<tr>
			<td>1.5 ml Microcentrifuge Tubes</td>
			<td>USA Scientific</td>
			<td>1415-2500</td>
			<td>500 tubes/bag</td>
		</tr>
		<tr>
			<td>15 ml conical screw cap centrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1475-0511</td>
			<td>25 tubes/bag</td>
		</tr>
		<tr>
			<td>50 ml conical screw cap centrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1500-1211</td>
			<td>25 tubes/bag</td>
		</tr>
		<tr>
			<td>Ni-NTA Superflow</td>
			<td>Qiagen</td>
			<td>30430</td>
			<td>NiNTA resin</td>
		</tr>
		<tr>
			<td>pH-indicator strips</td>
			<td>EMD Millipore Corporation</td>
			<td>1.09535.0001</td>
			<td>pH 0 - 14</td>
		</tr>
	</tbody>
</table>

modifying baculovirus expression vectors to produce secreted plant proteins in insect cells

Det har været en udfordring for forskerne at udtrykke rekombinant sekretoriske eukaryote proteiner til strukturelle og biokemiske undersøgelser. Baculovirus-medieret insekt celle udtryk system er et af de systemer, der bruges til at udtrykke rekombinant eukaryote sekretoriske proteiner med nogle posttranslationelle modifikationer. De sekretoriske proteiner skal dirigeres gennem de sekretoriske veje for protein glykosylering, disulfid obligationer dannelse og andre posttranslationelle modifikationer. For at forbedre den eksisterende insekt celle udtryk af sekretoriske vegetabilske proteiner, er et baculovirus udtryk vektor ændret ved tilsætning af enten en GP67 eller en hemolin signal peptid sekvens mellem arrangøren og kloning af flere websteder. Denne nydesignede modificerede vektorsystem produceret med succes et højt udbytte af opløselige rekombinant udskilles plante receptorproteiner fra Arabidopsis thaliana. To af udtrykt vegetabilske proteiner, de ekstracellulære domæner i Arabidopsis TDR og PRK3 plasmamembran receptorer, var krystalliseret til X-ray krystallografiske undersøgelser. Den modificerede vektorsystem er et forbedret værktøj, der kan potentielt bruges til udtryk af rekombinant sekretoriske proteiner i dyreriget så godt.

Som vist i figur 1, blev to modificerede pFastBac1 baculovirus udtryk vektorer brugt til at udtrykke de udskilles proteiner med enten GP67 eller hemolin signal sekvensen at erstatte iboende signal sekvensen af target-genet. Den virale GP67 og insekt hemolin gener er blevet påvist for at have høj sekretion udtryk niveauer i cellerne. Fusion proteiner med enten af disse to signal sekvenser forventes at have væsentligt forbedret sekretion udtryk niveauer. Ide...

Watch this Scientific Journal Video about Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells at JoVE.com

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

Ændre Baculovirus udtryk vektorer til at producere udskilles vegetabilske proteiner i insekt celler

Vi præsenterer her, protokoller for at udnytte den insekt celle og baculovirus protein udtryk system til at producere store mængder af planten udskilles protein til protein krystallisering. Et baculovirus udtryk vektor er blevet ændret med enten GP67 eller insekt hemolin signal peptid for plante protein sekretion udtryk i insekt celler.

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

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Biochemistry

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