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GenWay/FlashBAC - Baculovirus Expression System(24 reactions)/GWB-EB1FF8/Kit
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4000-520-616
Description:
Description | TheflashBACsystemisanewplatformtechnologyfortheproductionofrecombinantbaculoviruses.Mostimportantly,flashBAChasbeenspecificallydesignedtoremovetheneedtoseparaterecombinantvirusfromparentalvirusbyplaquepurificationoranyothermeans.Theproductionofrecombinantvirushasbeenreducedtoaone-stepprocedureininsectcellsandisthusfullyamenabletohighthroughputandautomatedproductionsystems.TheflashBACtechnologyhasbeendevelopedbythesameteamthatproducedthetriple-cut,linearDNA(BacPAK6)systemthathasbeenthestalwartofthebaculovirusexpressionsystemforthepast10years.AttheheartofflashBACtechnologyisanAcMNPVgenomethatlackspartofanessentialgene(ORF1629)andcontainsabacterialartificialchromosome(BAC)atthepolyhedringenelocus,replacingthepolyhedrincodingregion.TheessentialgenedeletionpreventsvirusreplicationwithininsectcellsbuttheBACallowstheviralDNAtobemaintainedandpropagated,asacirculargenome,withinbacterialcells.CircularviralDNAisthenisolatedfromthebacterialcellsandpurified.ThisistheflashBACDNAprovidedinthiskit.ArecombinantbaculovirusisproducedbysimplytransfectinginsectcellswithflashBACDNAandatransfervectorcontaining‘thegeneunderinvestigation’.Homologousrecombinationwithintheinsectcells(1)restoresthefunctionoftheessentialgeneallowingthevirusDNAtoreplicateandproducevirusparticlesand(2)simultaneouslyinserts‘thegeneunderinvestigation’underthecontrolofthepolyhedringenepromoterandremovestheBACsequence.Therecombinantvirusgenome,withtherestoredessentialgene,replicatestoproduceBVthatcanbeharvestedfromtheculturemediumofthetransfectedinsectcells(andformsaseedstockofrecombinantvirus).AsitisnotpossIBLefornon-recombinantvirustoreplicatethereisnoneedforanyselectionsystem..Thisone-stepproceduregreatlyfacilitatesthehighthroughputproductionofbaculovirusexpressionvectorsviaautomatedsystems.However,itisalsoofbenefittothesmallresearchgroupjustrequiringoneorafewrecombinantbaculovirusespreparedinindividualdishesofcells..TheflashBACsystemisbackcompatiblewithallbaculovirustransfervectorsbasedonhomologousrecombinationininsectcellsatthepolyhedringenelocus.Thisincludesvectorsusingthepolyhedrinpromoter,dual,tripleandquadrupleexpressionvectorsandthosethatuseothergenepromoterssuchasp10,ie1etc.ExamplesincludepBacPAK8/9,pAcUW31andpBacPAK-His1/2/3(BDBiosciencesClontech)butnotvectorssuchaspFastBac™,whicharedesignedforsite-specifictranspositioninE.coliusingtheBac-to-Bac®system(GibcoBRL).TheflashBACsystemalsomaximisesproteinsecretionandmembraneproteintargeting.Baculovirusgenomescontainseveralauxillarygenes,whicharenon-essentialforreplicationininsectcellculture.Oneoftheseischitinase(chiA),whichencodesanenzymewithexo-andendochitinaseactivity.Inaninfectedinsect,chitinase(togetherwithcathepsin)facilitateshostcuticlebreakdownandtissueliquefactionattheverylatestagesofinfection,soreleasingthevirustoinfectmorehosts.ConfocalandelectronmicroscopyobservationsofinsectcellsinfectedwithAcMNPVhaveshownthatchitinaseistargetedtotheendoplasmicreticulum(ER)whereitisdenselypackedinapara-crystallinearray,severelycompromisingthefunctionandefficacyofthesecretorypathway.DeletionofchiAfromflashBAChasimprovedtheefficacyofthesecretorypathwayandresultedinagreatlyenhanced(upto60-foldinsomeinstances)yieldofrecombinantproteinsthataresecretedormembranetargeted(incomparisonwithrecombinantvirusesthatsynthesisechitinase). |
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AdvantagesoftheflashBACsystem |
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AdditionalInformation:
Name | FlashBAC-BaculovirusExpressionSystem(24reactions) |
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RelatedProductNames | FlashBAC-BaculovirusExpressionSystem(24reactions) |
StABIlity | Guaranteed6months |
Storage | flashBACDNA:Storeat4°C.ControltransfervectorDNA(containinglacZreportergene):Storeat-20°C. |
IntendedUse | ResearchUseOnly |