Oxford Expression /flashBAC ULTRA/100304/3 reaction kit

Description:

flashBACULTRA™(cat#100304/100300/100301/100302) isthelatestdevelopmentintheseriesofflashBAC™vectors. InadditiontochiAandv-cath,threemorevirusgenes(p10,p74andp26)havebeendeletedfromtheflashBACULTRA™genome.Thishasallowedfurtherimprovementsinrecombinantproteinyieldandqualitytobedelivered,makingflashBACULTRAthebestchoiceforthemostdifficulttoexpressproteins.

UsingflashBAC™iseasierandquickerthanotherbaculovirusexpressionsystemsbecausethereisnoneedtoseparaterecombinantvirusfromparentalvirusbyplaque-purificationoranyothermeans;onlyrecombinantvirusisproducedaftertheco-transfection. Becausetheproductionofrecombinantvirushasbeenreducedtoaonestepprocedureininsectcells,itamenabletohighthroughputandautomatedproductionsystems. However,itisalsoofbenefittothesmallresearchgroupjustrequiringoneorafewrecombinantbaculovirusespreparedinindividualdishesofcells.

Baculovirusgenomescontainseveralauxillarygenes,whicharenon-essentialforreplicationininsectcellculture.Oneoftheseischitinase(chiA),whichencodesanenzymewithexo-andendochitinaseactivity.Inaninfectedinsect,chitinase,togetherwithserineproteasecathepsin,facilitateshostcuticlebreakdownandtissueliquefactionattheverylatestagesofinfection,soreleasingthevirustoinfectmorehosts.ConfocalandelectronmicroscopyobservationsofinsectcellsinfectedwithAcMNPVhaveshownthatchitinaseistargetedtotheendoplasmicreticulumwhereitisdenselypackedinaparacrystallinearray,severelycompromisingthefunctionandefficacyofthesecretorypathway.DeletionofchiAfromflashBAC™hasimprovedtheefficacyofthesecretorypathwayandresultedinagreatlyenhancedyieldofrecombinantproteins. Deletionofv-cathreducesthechancesofrecombinantproteindegradation.

Deletionofp10increasespolyhedrinpromoteractivityprovidingmorerecombinantprotein,increasesnuclearandcellularstABIlity,ensuringalongertimeframeforproteinexpressionandremovesamajorcompetitorforlimitingcellularresources.p74isnon-essentialincellculturebutisessentialfororalinfectivityofocclusion-derivedvirusinthehostwhereitplaysaroleinmidgutattachmentandfusion.Deletionofp74hasbeenshowntohavenoeffectonvirusproductioninvitro.Deletionofp74furtherincreasesthebiosafetyprofileofrecombinantbaculovirusesintheenvironment,makingthemunabletotraversetheinsectgutwall.P26isanearlygenethatcodesfora240-aminoacidpolypeptideofunknownfunctionandhasthesame5′terminusasp10.Deletionofthe3’-endofp26andfusiontolacZorp10havepreviouslybeenshowntohavenoeffectonvirusreplicationinvitro.Deletionofp10,p74andp26removesanunnecessarygeneticburdenfromtherecombinantvirusgenome,providingamoreefficientbaculovirusexpressionvector.