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Chapter8:Chapter8:ExpressionandModificationExpressionandModificationofRecombinantProteinsofRecombinantProteinsI.ProkaryoticexpressionsystemII.Eukaryoticexpressionsystem1.Yeastexpression2.Insectexpressionsystem3.MammalianexpressionsystemReferencesReferences1.Chapter11and13(EssentialsofMolecularBiology)2.Chapter10(AnIntroductiontoGeneticEngineering)I.ProkaryoticI.ProkaryoticExpressionSystemExpressionSystemTheredboxesrepresentexons,theblueboxesrepresenttheintrons,andthegreyboxesrepresentthe5’and3’UTRsInsertonlytheORF:intoanexpressionvectorthatcontainsprokaryotictranscriptionalandtranslationalregulatorysequencesHowdoyouexpressthisgeneinHowdoyouexpressthisgeneinbacteria?bacteria?SixStepProcessSixStepProcess①Isolationofgeneofinterest②Introductionofgenetoexpressionvector③Transformationintohostcells④Growthofcellsthroughfermentation⑤Isolation&purificationofprotein⑥FormulationofproteinproductCloningProcessCloningProcess*Geneofinterestiscutoutwithrestrictionenzymes(RE)*Hostplasmid(circularchromosome)iscutwithsameREs*Geneisinsertedintoplasmidandligatedwithligase*New(engineered)plasmidinsertedintobacterium(transform)FundamentalsofGeneFundamentalsofGeneExpressionExpression①Prokaryoticandeukaryoticpromotersandtranslationsignalsaredifferent...theyarenotexchangeableYouthereforecan’tsimplyputaeukaryoticpromoterintobacteriaandexpectittofunction②ProcessingalsopresentsaproblemforbacterialexpressionofhumanmRNAs.TheeukaryoticORFmustbeclonedintotheexpressionvector,notthegenomiccopy②ThegeneticcodeisidenticalformostbacterialandeukaryoticmRNAs.Althoughthecodeisthesame,expressionlevelscanbeaffectedbycodonfrequency,whichvariesbetweenorganismsandtranscripts④Post-translationalmodificationscanbeimportantforproteinfunctionThosemodificationsmightnotoccurinbacteria.Thesolution…tryexpressinginaeukaryoticexpressionsystem(viral,baculovirus,yeast)ProkaryoticExpressionProkaryoticExpressionPros*Convenient/Easy*Produceandpurifyproteinusingtheleastexpensiveandeasiestreagentsandequipment*BestforlargescaleproductionofproteinCons*lackmanyoftheimmunogenicproperties*3D“Native”conformation*LackPTMs(Posttranslationalmodifications)neededforspecificactivityWhichVector?WhichVector?Promoters*arabinosesystems(pBAD),phageT7(pET),Trc/Tacpromoters,λPLorPRTags标签*His6formetalaffinitychromatography(Ni)*FLAGepitopetageDYKDDDDK*CBP-calmodulinbindingpeptide(26residues)*E-coil/K-coiltags(polyE35orpolyK35)*c-mycepitopetagEQKLISEEDL*Glutathione-S-transferase(GST)tags*Celluluosebindingdomain(CBD)tagsT7PromoterlacOperatorRBSATGTAGpeptidetag:V5/poly-(his)peptidetag:V5/poly-(his)mutiplecloningsite(mcs)Gene(with/withoutstopcodon)FusionProteinFusionProteinAGenericVectorII.EukaryoticExpressionII.EukaryoticExpressionSystemSystem1.Yeastexpression2.Insectexpressionsystem3.Mammalianexpressionsystem1.YeastExpression1.YeastExpression􀂄Pros*Easierandlessexpensivethanhighereukaryoticcells*Rapidgrowthoninexpensivemedia*Idealforlarge-scaleproductionofheterologousproteins*Oftencomfortablewithgeneticmanipulation*Exhibitnear-nativeconformation,PTMs,processing*Well-definedsecretorypathwaysforextracellularexportoftherecombinantgeneproduct*UsuallysafetouseCons*LacksomePTMsrequiredforspecificactivities*Alittlemoreexpensivethanprokaryotesystems*OftenlackmechanismsforproperfoldingforsomeeukaryoteproteinsYeastExpressionVectorPichiapastorisComm...