生物催化与生物转化V

Production,IsolationandPurificationoftheFunctionalProteinorPolypeptideProteinorPolypeptideInsulin(diabetes)Interferonb(relapsingMS)Interferong(granulomatous)TPA(heartattack)Actimmune(Ifg)Activase(TPA)BeneFix(FIX)Betaseron(Ifb)HumulinNovolinPegademase(AD)EpogenRegranex(PDGF)Novoseven(FVIIa)Intron-ANeupogenPulmozymeInfergenProteinorPolypeptideFirst“proteinvaccine〞wascow-pox(Jenner,1796)Firstproteinpharmaceuticalwasinsulin(Banting&Best,1922)Nowmorethan200approvedpeptideandproteinpharmaceuticalsontheFDAlist()Manydifferentsources…ProteinPharmaceuticalsInsulin Pigsorcattle(pancreas)Albumin Humanblood(donated)HGH HumanbrainsFactorVIII Humanblood(donated)Calcitonin SalmonAnti-venom HorseofGoatbloodProteinDrug

OriginalSourceProteinPharmaceuticalsFromBloodBodycontains6litresofblood60-70%ofbloodisplasma,8-9%isprotein–apharmaceuticalcornucopiaPlasmacontains10,000differentproteins(~20proteinsmakeup99%ofplasmaproteins)Discarded,donatedblood~2millionlitres/year–greatsourceforproteinsBloodFractionationKeyBloodProductsFactorVIII(forbloodclotting-hemophilia)FactorIX(forbloodclotting-hemophilia)Albumin(osmoticbalance-kidneydisease)IgIV(fortreatinginfections)Anti-thrombinIII(forbloodclotting)AlphaI-PI(foremphysema,AIAD)AllpreparedbyCohnFractionation(1946)Differentialprecipitationbyethanol,salt,pH,temperature,centrifugationPeptideDrugsManyhormonesareactuallysmallpeptides(2-40aminoacids)Calcitonin(Calcimar,Miacalcin,32res.)ThyroidhormonetoenhancebonemassOxytocin(Pitocin,9residues)PituitaryhormonetostimulatelaborVasopressin(Pitressin,9residues)Pituitaryh.forantidiuretic/vasconstrictionAutomaticPeptideSynthesizer(ABI433A)ProteinPharmaceuticalsNaturalsourcesareoftenrareandexpensiveDifficulttokeepupwithdemandHardtoisolateproductLeadtoimmunereactions(diff.species)Viral&pathogencontaminationMostproteinpharmaceuticalstodayareproducedrecombinantlyCheaper,safer,abundantsupplyRecombinantMethodsDevelopedin1970’s&1980’sPaulBerg(1973)restrictionenzymesHerbertBoyer(1978)cloninghumaninsulinintoE.coli–GenentechTwogeneralapproachesExpressioninisolatedcellsExpressionintransgenicplants/animalsSixStepProcessIsolationofgeneofinterestIntroductionofgenetoexpressionvectorTransformationintohostcellsGrowthofcellsthroughfermentationIsolation&purificationofproteinFormulationofproteinproductCloningProcessGeneofinterestiscutoutwithrestrictionenzymes(RE)Hostplasmid(circularchromosome)iscutwithsameREsGeneisinsertedintoplasmidandligatedwithligaseNew(engineered)plasmidinsertedintobacterium(transform)Cloning(Details)Cloning(Details)proteinRecombinantProteinExpressionSystemsEscherichiacoliOtherbacteriaPichiapastorisOtheryeastBaculovirusAnimalcellculturePlantsSheep/cows/humansCellfreePolyhedraExpressionSystemSelectionChoicedependsonsizeandcharacterofproteinLargeproteins(>100kD)?ChooseeukaryoteSmallproteins(<30kD)?ChooseprokaryoteGlycosylationessential?ChoosebaculovirusormammaliancellcultureHighyields,lowcost?ChooseE.coliPost-translationalmodificationsessential?Chooseyeast,baculovirusorothereukaryoteWhichVector?Mustbecompatiblewithhostcellsystem(prokaryoticvectorsforprokaryoticcells,eukaryoticvectorsforeukaryoticcells)Needsagoodcombinationofstrongpromotersribosomebindingsitesterminationsequencesaffinitytagorsolubilizationsequencesmulti-enzymerestrictionsitePlasmidsandVectorsCircularpiecesofDNAranginginsizefrom1000to10,000basesAbletoindependentlyreplicateandtypicallycodefor1-10genesOftenderivedfrombacterial“mini〞chromosomes(usedinbacterialsex)Mayexistassinglecopiesordozensofcopies(oftenusedtotransferantibioticresistance)KeyPartstoaVectorOriginofreplication(ORI)–DNAsequenceforDNApolymerasetoreplicatetheplasmidSelectablemarker(AmporTet)–agene,whenexpressedonplasmidwillallowhostcellstosurviveInduciblepromoter–ShortDNAsequencewhichenhancesexpressionofadjacentgeneMulti-cloningsite(MCS)–ShortDNAsequencethatcontainsmanyrestrictionenzymesitesAGenericVectorWhichVector?Promotersarabinosesystems(pBAD),phageT7(pET),Trc/Tacpromoters,phagelambdaPLorPRTagsHis6formetalaffinitychromatography(Ni)FLAGepitopetageDYKDDDDKCBP-calmodulinbindingpeptide(26residues)E-coil/K-coiltags(polyE35orpolyK35)c-mycepitopetagEQKLISEEDLGlutathione-S-transferase(GST)tagsCelluluosebindingdomain(CBD)tagsGeneIntroduction(Bacteria)BacterialTransformationBacterialTransformationMovestheplasmidintobacterialhostEssentialtomakingthegene“actively〞expresstheproteininsidethecell2routesoftransformationCaCl2+coldshockElectroporationTypicaltransformationrateis1in10,000cells(notveryefficient)forCaCl2,but1in100forelectroporationElectroporator25microfarads=2500V@200ohmsfor5msElectroporationSeemstocausedisruptionincellmembraneReconstitutionofmembraneleadstolargeporeswhichallowDNAmoleculestoenterWorksforbacteria,yeastandanimalcellsBacterialSystemsGrowquickly(8hrstoproduceprotein)Highyields(50-500mg/L)Lowcostofmedia(simplemediaconstituents)LowfermentorcostsDifficultyexpressinglargeproteins(>50kD)NoglycosylationorsignalpeptideremovalEukaryoticproteinsaresometimestoxicCan’thandleS-Srichproteins

Advantages

DisadvantagesCloning&TransforminginYeastCellsPichiapastorisPichiaPastorisYeastaresinglecelledeukaryotesBehavelikebacteria,buthavekeyadvantagesofeukaryotesP.pastorisisamethylotrophicyeastthatcanusemethanolasitssolecarbonsource(usingalcoholoxidase)Hasaverystrongpromoterforthealcoholoxidase(AOX)gene(~30%ofproteinproducedwheninduced)PichiaCloningPichiaPastorisCloningUsesaspecialplasmidthatworksbothinE.coliandYeastOncegeneofinterestisinsertedintothisplasmid,itmustbelinearized(cutopensoitisn’tcircular)Doublecross-overrecombinationeventoccurstocausethegeneofinteresttoinsertdirectlyintoP.pastorischromosomewheretheoldAOXgeneusedtobeNowgeneofinterestisundercontrolofthepowerfulAOXpromoterPichiaSystemsGrowquickly(8hrstoproduceprotein)Veryhighyields(50-5000mg/L)Lowcostofmedia(simplemediaconstituents)LowfermentorcostsCanexpresslargeproteins(>50kD)Glycosylation&signalpeptideremovalHaschaperoninstohelpfold“tough〞prtnsCanhandleS-Srichproteins

Advantages

MoreadvantagesBaculovirusExpressionBaculovirusExpressionAutographicacalifornicamultiplenuclearpolyhedrosisvirus(Baculoviurs)Viruscommonlyinfectsinsectscellsofthealfalfalooper(smallbeetle)orarmyworms(andtheirlarvae)Usessuper-strongpromoterfromthepolyhedroncoatproteintoenhanceexpressionofproteinswhilevirusresidesinsidetheinsectcellBaculovirusExpression~12daysBaculovirus(AcMNPV)CloningProcess5’3’TransfervectorPolyhedringenexxClonedgeneAcMNPVDNA5’3’ClonedgeneRecombinantAcMNPVDNABaculovirusSuccessesAlphaandbetainterferonAdenosinedeaminaseErythropoietinInterleukin2PoliovirusproteinsTissueplamsinogenactivator(TPA)BaculovirusSystemsGrowveryslowly(10-12daysforset-up)Cellcultureisonlysustainablefor4-5daysSet-upistimeconsuming,notassimpleasyeastCanexpresslargeproteins(>50kD)Correctglycosylation&signalpeptideremovalHaschaperoninstohelpfold“tough〞prtnsVeryhighyields,cheap

Disadvantages

AdvantagesMammalianExpressionSystemsMammalianCell-lineExpressionSometimesrequiredfordifficult-to-expressproteinsorfor“completeauthenticity〞(matchingglycosylationandsequence)CellsaretypicallyderivedfromtheChineseHamsterOvary(CHO)celllineVectorsusuallyuseSV-40virus,CMVorvacciniaviruspromotersandDHFR(dihydrofolatereductase)astheselectablemarkergeneMammalianExpressionGeneinitiallyclonedandplasmidpropagatedinbacterialcellsMammaliancellstransformedbyelectroporation(withlinearplasmid)andgeneintegrates(1ormoretimes)intorandomlocationswithindifferentCHOchromosomesMultipleroundsofgrowthandselectionusingmethotrexatetoselectforthosecellswithhighestexpression&integrationofDHFRandthegeneofinterestMethotrexate(MTX)SelectionGeneofinterestDHFRTransfectdfhr-cellsGrowinNucleosideFreemediumCultureaColonyofcellsGrowin0.05uMMtxCultureaColonyofcellsMethotrexate(MTX)SelectionGrowin5.0uMMtxGrowin0.25uMMtxCultureaColonyofcellsCultureaColonyofcellsForeigngeneexpressedinhighlevelinCHOcellsMammalianSystemsSelectiontakestime(weeksforset-up)CellcultureisonlysustainableforlimitedperiodoftimeSet-upisverytimeconsuming,costly,modestyieldsCanexpresslargeproteins(>50kD)Correctglycosylation&signalpeptideremoval,generatesauthenticproteinsHaschaperoninstohelpfold“tough〞prtns

Disadvantages

AdvantagesMammalianCellSuccessesFactorIXFactorVIIIGammainterferonInterleukin2HumangrowthhormoneTissueplamsinogenactivator(TPA)ConclusionIsolationofgeneofinterestIntroductionofgenetoexpressionvectorTransformationintohostcellsGrowthofcellsthroughfermentationIsolation&purificationofproteinFormulationofproteinproductCellGrowthNeedsAsterilecarbon,nitrogen,hydrogenandoxygensource(airandH2O)+tracemetals(Zn,Fe,Cu,Ca,Mg,Mn)Asterileenergysource(light,sugar,acetate,methanol,ethanol)Aconstant(ornearconstant)temperatureabove20oCAgrowthregulatingchemical(antibiotic)Prototrophsvs.AuxotrophsProtrophiccells(bacteria,plants)canproduceallessentialaminoacids,nucleicacids,carbohydratesandlipidsfromsimplenutrients(water,oxygen,nitrogenorammonia,CO2)Auxotrophiccells(yeast,insectcells,mammalian)needvitamins,essentialaminoacids(His,Cys),sugars,lipids,etc.togrowbecausetheyhavelostthisabilitythroughevolution(bacterialsymbiosis)FeaturesMicrobesAnimalCellsCellwallGenerallypresentGenerallyabsentCellmembranePresentPresentGrowthRate10-50%perhour1-5%perhourO2RequirementHighLowNutritionalRqmtUsuallysimpleComplexCO2RequirementSometimesKeyforbufferingEnvironmentalFXLessaffectedVerysusceptibleSize100-2000nm10000-100000nmSeedingdensity1cell105

cells/mLGrowthdensity109-1010

cells/mL106

cells/mLAllCellsCanbeGrowninIncubatorsorFermentorsShakeFlaskIncubatorShakeFlaskIncubatorG25NewBrunswickFloorModelIncubatorCutawayModelIncubatorShakeFlaskIncubatorsSometimescalledenvironmentalchambersHeavilyinsulated,heatedwiththermoregulationtokeeptemperaturewithin0.5oCofset-pt.Rotatableplatformtospinupto500rpmtofacilitateaeration(dissolvesN2andO2neededforgrowth)Designedforsmall-scalegrowthFermentors&BioreactorsLargerscale,sustainedgrowthrequiresbioreactors&fermentorsFermentorshavebeenusedforcenturies–primarilyforbrewingalcoholandmakingvinegarModerntechnologyandchemicalengineeringprinciplescontinuetoimprovefermentordesignDateProductsVesselsProcessControlCultureMethodQualitycontrol3000BCto1900AlcoholVinegarWoodenCopperTraysThermometerheatexchangerBatchMethodsNil1900to1940GlycerolCitricacidAcetoneSteelwithmechanicalstirringpHcontrolTempcontrolBatchandFed-BatchAlmostNil1940topresentAntibioticsNucleotidesAminoacidsSterilemechanicalaeratedpH&O2electrodesTempcontrolFed-BatchandcontinuousVeryimportant1960topresentNativeproteins&enzymesPressurejetvesselsComputerlinkedcontrolloopsContinuousculturewithrecyclerVeryimportant1979topresentRecombinantproteins&enzymesFermentors(all4types)ComputerlinkedcontrolpH,O2,TempBatch,FedBatch&ContinuousVeryimportantFermentors&BioreactorsFourbasicbioreactordesignsStirredtankreactors(mechanicalagitationforaeration)Bubblecolumnreactors(bubblingairintomediaforaeration)Internalloopairliftreactors(airandmediacirculatetogether)ExternalloopairliftreactorsStirredTankFermentor/BioreactorFourBioreactorDesignsAirliftReactorsStirredTankReactorFermentorScaleUpCan’tstartcellculturein100000L,mustdorepeated,scaledinoculationsStartwithstockculture(5-10mL)Thenshakerflask(200-500mL)Thenseedfermentor(10Lto100L)Thenproductionfermentor(1000Lto100,000L)CellIsolation/HarvestingCellSuspensionCellConcentrateCellsCell-freeculturemediumMembraneCrossFlowFiltrationCell-freeculturemediumDeadEndFiltrationProteinIsolation&PurificationAftercells(ormedia)areharvestedproteinsmaybepurified/isolatedIntracellular(insidecell)proteinsarehardertopurifyRequirecelldisruption,separation,removalofcelldebris,DNA,RNA,lipidExtracellular(outsidecell)proteinsareeasiertopurifyNocelldisruptionneeded,justisolateCellDisruptionMethodsSonicationFrenchpressGlassbeaddisruptionEnzymaticlysisDetergentlysisFreeze-thawOsmoticlysisGentleMethodsVigorousMethodsProteinIsolationMethodsDifferentialsaltprecipitationDifferentialsolventprecipitationDifferentialtemperatureprecipitationDifferentialpHprecipitationTwo-phasesolventextraction(PEG)PreparativeelectrophoresisColumnchromatographyMostpurificationsrequirecombinationsof2-3stepsElectrophoresisElectrophoresisPrincipleistoseparateproteins(intact)onthebasisoftheirchargeandtheirabilitytomigratewithinagel(jello-like)matrixAstrongelectricfieldisappliedtotheproteinmixtureforanextendedperiodoftime(hours)untiltheproteinsmoveapartormigrateIsoelectricFocusing(IEF)IEFPrinciplesANODE+++++++++_________CATHODEIncreasingpHpI=8.6pI=6.4pI=5.1IsoelectricFocusingSeparationofbasisofpI,notMwRequiresveryhighvoltages(5000V)Requiresalongperiodoftime(10h)PresenceofapHgradientiscriticalDegreeofresolutiondeterminedbyslopeofpHgradientandelectricfieldstrengthKeepsproteinstructureintactCanbescaleduptoisolatemgtogmsofproteininasingle“tube〞gelrunColumnChromatographyColumnChromatographyMostcommon(andbest)approachtopurifyinglargeramountsofproteinsAbletoachievethehighestlevelofpurityandlargestamountofproteinwithleastamountofeffortandthelowestlikelihoodofdamagetotheproteinproductStandardmethodforpharmaindustryColumnChromatographyCanbedoneeitheratatmosphericpressure(gravityfeed)orathighpressure(HPLC,500-2000psi)Fourtypesofchromatography:AffinitychromatographyGelfiltration(sizeexclusion)chrom.IonexchangechromatographyHydrophobic(reversephase)chrom.AffinityChromatographyAdsorptiveseparationinwhichthemoleculetobepurifiedspecificallyandreversiblybinds(adsorbs)toacomplementarybindingsubstand(aligand)immobilizedonaninsolublesupport(amatrixorresin)Purificationis1000Xorbetterfromasinglestep(highestofallmethods)PreferredmethodifpossibleAffinityChromatographyStep1:AttachligandtocolumnmatrixStep2:LoadproteinmixtureontocolumnAffinityChromatography

Step3:ProteinsbindtoligandsStep4:Washcolumntoremoveunwantedmaterial,elutelaterAffinityChromatographyUsedinmanyapplicationsPurificationofsubstancesfromcomplexbiologicalmixturesSeparationofnativefromdenaturedformsofproteinsRemovalofsmallamountsofbiomaterialfromlargeamountsofcontaminantsAffinityChromatographyTheligandmustbereadily(andcheaply)availableLigandmustbeattachable(covalently)tothematrix(typicallysepharose)suchthatitstillretainsaffinityforproteinBindingmustnotbetoostrongorweakElutioninvolvespassageofhighsaltorlowpHbufferafterbindingLigandSpecificityAMPEnzymeswithNADcofactorsanATPdependentkinasesArginineProteasessuchasprothrombin,kallikrein,clostripainCibacronBlueDyeSerumAlbumin,PreabluminHeparinGrowthfactors,cytokines,coagulationfactorsProteinAFcregionofimmunoglobulinsCalmodulinCalmodulinregulatedkinases,cylcasesandphosphatasesEGTA-copperProteinswithpoly-HistidinetailsSizeExclusionChrom.MoleculesareseparatedaccordingtodifferencesintheirsizeastheypassthroughahydrophilicpolymerPolymerbeadscomposedofcross-linkeddextran(dextrose