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 arbeidet ble støttet av de nye fakultetet oppstart midlene fra North Carolina State University for Guozhou Xu.

Merk: Et insekt cellen/baculovirus system med endrede uttrykk vektorer for sekretoriske plante protein uttrykk og krystallisering brukes.
1. endring av en Baculovirus uttrykk vektor med GP67 Signal peptid plante Protein sekresjon uttrykk
<ol><li>Syntetisere et DNA fragment som inneholder en 5' BglII kutte området13, GP67 sekresjon signal sekvensen og en multi-kloning området med NotI, BamHI, EcoRI, StuI, SalI, SpeI, XbaI, PstI og XhoI (figur 1A<...

<ol>
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Gitt mangfoldet i størrelse og stabilitet av tusenvis av proteiner tilstede i biologiske systemer, er det ofte empirisk for en forskning laboratorium bestemme hvilket heterologous uttrykk system som skal velges for uttrykk for et bestemt protein. E. coli uttrykk systemet er ofte førstevalget for protein uttrykk på grunn av den korte livssyklusen for bakterier, lavpris kultur medier og relativt lett å skalere opp19. For uttrykk for store eukaryote proteiner med størrelse mer enn 60 kD...

Det er viktig for et forskningslaboratorium skal produsere store mengder homogen rekombinante proteinene for biokjemisk og Biofysiske karakteristikk, spesielt for X-ray krystallografisk studier. Det er mange godt etablerte heterologous uttrykk systemer som Escherichia coli, gjær, insekt celler, pattedyrceller, anlegg celler, etc. blant dem, baculovirus-mediert insekt cellen uttrykk systemet er en av mest brukte teknikker til å produsere store mengder strukturelt foldet store rekombinant eukaryote proteiner for protein krystallisering1.
Uttrykket vektorer av baculovirus uttrykk er konstruert for å inneholde en sterk polyhedrin eller P10 arrangøren å produsere en høy avkastning rekombinant intracellulær proteiner2,3. For å gjøre en rekombinant baculovirus, er genet av interesse klonet i et insekt vektor som inneholder polyhedrin (polh) locus Autographa californica multi-nucleopolyhedroviral genomet. Den resulterende konstruere er deretter sekvensielt og åpen leses rammen (ORF) er kontrollert. Den riktige konstruere er så introdusert i vert insekt cellen gjennom prosessen med hva. Genet av interesse settes inn i viral genomet av homologe rekombinasjon. Denne hendelsen resulterer i produksjon av rekombinant viral genomet, som deretter gjentak å produsere rekombinant okuleres virus partikler1.
I insekt celler som vanligvis brukes i uttrykk systemet er Sf9 og høy fem (Hi5) celler. Sf9 celler er en klonal isolere av Sf21, utledet fra pupal ovarian cellene i Spodoptera frugiperda, Hi5 cellene er en klonal isolere stammer fra det foreldre Trichoplusia ni ovarian celle linje TN-3684,5. Co transfections, virus forsterkning og plakk analyser er gjennomført på Sf9 celler, mens Hi5 cellene er vanligvis valgt å produsere høyere mengder rekombinante proteinene6. Det er verdt å merke seg at Hi5 cellene ikke er egnet for generasjon og forsterkning av virus progenies hensyn til deres tendensen å produsere mutant virus. Tradisjonelt anses et temperaturområde på 25-30 ° C å være bra for dyrking av insekt celler. Det har imidlertid blitt rapportert at 27-28 ° C er den optimale temperaturen for insekt celle vekst og infeksjon7,8.
Innføring av et sterkt signal før genet er nødvendig for høy uttrykk for secreted proteiner. Signalet sekvensen ville effektivt guide oversatt rekombinant protein i det endoplasmatiske retikulum protein sekresjon og post-translasjonell modifikasjoner nødvendig for riktig sammenleggbar og stabilisering3. Signalet peptid sekvenser, har slik som baculovirus innhylle protein GP64/67, honeybee melittin og andre, blitt valgt å forenkle uttrykk for sekretoriske rekombinante proteinene i baculovirus-mediert uttrykk systemer3. Innføring av signal peptid av GP67 har vist seg å forbedre uttrykk avkastningen av en secreted rekombinant protein, i forhold til bruker den iboende signal peptid av målet genet9. Hemolin er et hemolymph protein av gigantiske silke møll Hyalophora cecropia, indusert på bakterier smitte10. På grunn av den relativt høye nivået av indusert uttrykk, kan signalet peptid sekvensen av genet brukes å megle sekresjon uttrykk for rekombinante proteinene i baculovirus-insekt cellen systemet.
A. thaliana Tracheary Element differensiering hemmende faktor reseptor (TDR) og Pollen reseptor Kinase 3 (PRK3) begge tilhører anlegget Leucine-rik gjenta reseptor-lignende Kinase (LRR-RLK) familie av proteiner11,12 . For å studere struktur og funksjon av denne familien av anlegget reseptor proteiner, samt å lette strukturelle og biokjemiske karakterisering av andre plante utskilles proteiner, har baculovirus-insekt cellen uttrykk systemet blitt endret til forbedre protein kvalitet og dekkevne. Ekstracellulære domenene TDR og PRK3 blitt er uttrykt bruke to endrede uttrykk vektorer i baculovirus-insekt cellen uttrykk systemet. Begge ekstracellulære domenene TDR og PRK3 proteiner har blitt krystallisert. Denne artikkelen viser uttrykk og rensing av store mengder rekombinant utskilles anlegget proteiner med to modifisert baculovirus uttrykk vektorer ved å innlemme en GP67 eller en hemolin signal sekvens mellom selskapet og flere kloning nettsteder.

<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 vært en utfordring for forskere å uttrykke rekombinant sekretoriske eukaryote proteiner for strukturelle og biokjemiske studier. Baculovirus-mediert insekt cellen uttrykk systemet er en av å uttrykke rekombinant eukaryote sekretoriske proteiner med noen post-translasjonell modifikasjoner. Sekretoriske proteiner må rutes gjennom sekretoriske veier for protein glykosylering, disulfide obligasjoner dannelse og andre post-translasjonell endringer. For å forbedre eksisterende insekt celle uttrykket av sekretoriske anlegget proteiner, er en baculovirus uttrykk vektor endret av tillegg av enten et GP67 eller en hemolin signal peptid sekvens mellom selskapet og flere-kloning nettsteder. Denne nydesignede endret vektor-system produsert en høy avkastning av løselig rekombinant utskilles anlegget reseptor proteiner av Arabidopsis thaliana. To av uttrykt anlegget proteiner, ekstracellulære domener av Arabidopsis TDR og PRK3 plasma membran reseptorer, ble krystallisert for X-ray krystallografisk studier. Modifisert vektor systemet er et forbedret verktøy som kan potensielt brukes for uttrykket av rekombinant sekretoriske proteiner i dyreriket også.

Som vist i figur 1, ble to modifisert pFastBac1 baculovirus uttrykk vektorer brukt til å uttrykke secreted proteiner i GP67 eller hemolin signal sekvensen erstatte iboende signal sekvensen av målet genet. Viral GP67 og insekt hemolin genene har vist seg for å ha høy sekresjon uttrykk nivåer i cellene. Fusion proteiner med en av disse to signal sekvenser forventes å har forbedret sekresjon uttrykk nivåer. Identisk flere kloning områder (MCS) ble konstr...

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

Endre Baculovirus uttrykk vektorer å produsere utskilles anlegget proteiner i insekt celler

Her presenterer vi protokollene for utnytte insekt celler og baculovirus protein uttrykk systemet å produsere store mengder anlegget utskilles proteiner for protein krystallisering. En baculovirus uttrykk vektor er endret med GP67 eller insekt hemolin signal peptid plante protein sekresjon uttrykk 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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