昆虫杆状病毒细胞表达系统-课件

5.2.2—5.2.2—••••„„„„„„„„„„„„1)Functionalactivityoftherecombinantprotein„TheBEVStypicallyproducesoverexpressedrecombinantproteinscontainingproperfolding,disulfidebondformationandoligomerization.Additionally,thissystemiscapableofperformingseveralpost-translationalmodifications.Thisleadstoaproteinthatissimilartoitsnativecounterpart,bothstructurallyandfunctionally.However,incaseswheretheauthenticproteinfunctionsasaheterodimerorreliesontissue-orspecies-specificmodifications,therecombinantBaculovirus-expressedproteinmaynotbefunctionallyactive,unlessitsbindingpartnerormodifyingenzymeisco-expressed.2)Post-translationalmodifications2)Post-translationalmodifications„Severalpost-translationalmodificationshavebeenreportedtooccurintheBEVS,includingN-andO-linkedglycosylation,phosphorylation,acylation,amidation,carboxymethylation,isoprenylation,signalpeptidecleavageandproteolyticcleavage.Thesiteswherethesemodificationsoccurareoftenidenticaltothoseoftheauthenticproteininitsnativecellularenvironment.„Severalpost-translationalmodificationshavebeenreportedtooccurintheBEVS,includingN-andO-linkedglycosylation,phosphorylation,acylation,amidation,carboxymethylation,isoprenylation,signalpeptidecleavageandproteolyticcleavage.Thesiteswherethesemodificationsoccurareoftenidenticaltothoseoftheauthenticproteininitsnativecellularenvironment.3)Highexpressionlevels3)Highexpressionlevels„Comparedtootherhighereukaryoticexpressionsystems,themostdistinguishingfeatureoftheBEVSisitspotentialtoachievehighlevelsofexpressionofaclonedgene.TheBEVSsystemhasprovenparticularlyusefulinthegenerationoflargequantitiesofproteinsforstructuralanalysis.„Thehighestexpressionlevelreportedis50%ofthetotalcellularproteinofaninfectedinsectcellcorrespondingtoapproximately1gofrecombinantproteinper1109cells.However,manyrecombinantproteinsarenotproducedatsuchhighamountsanditisusuallydifficulttopredicttheamountofproteinexpression.Therearesomeguidelinesonecanfollowtooptimizeproteinproduction.OfprimaryimportanceisoptimizingthedesignoftherecombinantBaculovirusTransferVector.„Comparedtootherhighereukaryoticexpressionsystems,themostdistinguishingfeatureoftheBEVSisitspotentialtoachievehighlevelsofexpressionofaclonedgene.TheBEVSsystemhasprovenparticularlyusefulinthegenerationoflargequantitiesofproteinsforstructuralanalysis.„Thehighestexpressionlevelreportedis50%ofthetotalcellularproteinofaninfectedinsectcellcorrespondingtoapproximately1gofrecombinantproteinper1109cells.However,manyrecombinantproteinsarenotproducedatsuchhighamountsanditisusuallydifficulttopredicttheamountofproteinexpression.Therearesomeguidelinesonecanfollowtooptimizeproteinproduction.OfprimaryimportanceisoptimizingthedesignoftherecombinantBaculovirusTransferVector.4)Capacityforlargeinserts4)Capacityforlargeinserts„TheexpandabilityofthecapsidstructureofBaculovirusesallowsthepackagingandexpressionofverylargegenes.ThereisnoknownuppersizelimitfortheinsertionofforeignsequencesintotheBVgenome.„TheexpandabilityofthecapsidstructureofBaculovirusesallowsthepackagingandexpressionofverylargegenes.ThereisnoknownuppersizelimitfortheinsertionofforeignsequencesintotheBVgenome.5)Capacitytoexpressunsplicedgenes5)Capacitytoexpressunsplicedgenes„Insectcellshavethecapabilitytoperformintron/exonsplicing.However,certainvirus-,tissue-orspecies-specificsplicingpatternswillnotbeobtainediftheyrequirethepresenceofparticularsplicingfactorswhicharenotavailableintheinfectedinsectcellenvironment.Ingeneral,forhighproteinexpressionlevels,acDNAinsertratherthanagenomicDNAfragmentisrecommended.„Insectcellshavethecapabilitytoperformintron/exonsplicing.However,certainvirus-,tissue-orspecies-specificsplicingpatternswillnotbeobtainediftheyrequirethepresenceofparticularsplicingfactorswhicharenotavailableintheinfectedinsectcellenvironment.Ingeneral,forhighproteinexpressionlevels,acDNAinsertratherthanagenomicDNAfragmentisrecommended.6)Simplicityoftechnology6)Simplicityoftechnology„Thistechnologyhasmadeexpressionoffull-lengthproteinsfast,easyandreliable.RecombinantBaculoviruscanbeobtainedintwosimplesteps–cloningandco-transfection–inaslittleas5days.TheeaseofusenowrivalsthatofbacterialexpressionsystemsandBEVStechnologydoesnotrequirethattherecombinantproteinbeexpressedasafusionprotein.„Thistechnologyhasmadeexpressionoffull-lengthproteinsfast,easyandreliable.RecombinantBaculoviruscanbeobtainedintwosimplesteps–cloningandco-transfection–inaslittleas5days.TheeaseofusenowrivalsthatofbacterialexpressionsystemsandBEVStechnologydoesnotrequirethattherecombinantproteinbeexpressedasafusionprotein.7)Simultaneousexpressionofmultiplegenes7)Simultaneousexpressionofmultiplegenes„BEVShasthecapabilitytoexpresstwoormoregenessimultaneouslywithinsingleinfectedinsectcells.Proteincomplexesthatdependondimerormultidimerformationforactivitycanbeassembled.Awellknownexampleistheformationofcompleteviruscapsidsfromavarietyofviruseswhichhavebeenassembledinvitro,usingBEVS,bycoexpressingthecapsidsubunitssimultaneously.„BEVShasthecapabilitytoexpresstwoormoregenessimultaneouslywithinsingleinfe