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).
| AdvantagesoftheflashBACsystem | •Simpletouse •Onestepproductionofrecombinantvirusininsectcells •Nostepsneededtopurifyrecombinantvirus •Amenabletohighthroughputandautomatedsystems •Maximisesproductionofsecretedandmembrane-targetedproteins •Back-compatiblewithahugerangeoftransfervectors. |
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AdditionalInformation:
| Name | FlashBAC-BaculovirusExpressionSystem(5reactions) |
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| RelatedProductNames | FlashBAC-BaculovirusExpressionSystem(5reactions) |
| StABIlity | Guaranteed6months |
| Storage | flashBACDNA:Storeat4°C.ControltransfervectorDNA(containinglacZreportergene):Storeat-20°C. |
| IntendedUse | ResearchUseOnly |
