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高效蛋白表达系统第1页,共65页,2023年,2月20日,星期四ProteinExpressionProteinexpressionisasubcomponentofgeneexpression.ItconsistsofthestagesafterDNAhasbeentranslatedintoaminoacidchains,whichareultimatelyfoldedintoproteins.Initssimplestform,aproteinexpressionsysteminvolvesatemplate,amechanismoftranscription/translationsuchasacellorcellextract,andtherawmaterialsrequiredtobuildproteins.Proteinexpressioncanbedoneinprokaryotesiftheproteindoesn’tneedtobepost-translationallymodified.Ifperformingstructure-functionstudies,expressinginaeukaryoticsystemisimportantbecausethesecellshavethepropercellularmachinerytodecoratetheproteininquestionwiththecorrectpost-translationalmodifications.Thetypesofeukaryoticcellstypicallyusedinproteinexpressionincludeyeast,insect,andmammalian.第2页,共65页,2023年,2月20日,星期四ChoiceoftheexpressionsystemCell-freeBacteriaYeastInsectMammalianEasyofuseCostofmediaandEquipmentPos-translationalModifications(Probabilityofproteinfunction)TimeRequirement第3页,共65页,2023年,2月20日,星期四ProkaryoticExpression第4页,共65页,2023年,2月20日,星期四ProteinexpressioninprokaryoticsystemBacterialexpressionvectorshavesomedistinctfeatures:

Induciblepromotersystems;Proteinfusionsincludingfusedtags;Easymanipulation;第5页,共65页,2023年,2月20日,星期四SomeproblemsofproductioninE.coli

第6页,共65页,2023年,2月20日,星期四Inclusionbodies(mostcommoncase)

Inclusionbodiesareformed

throughtheaccumulationoffoldingintermediates

ratherthanfromthenativeorunfoldedproteins.

Itisnotpossibletopredictwhichproteinswillbeproducedasinclusionbodies.

Productionofinclusionbodiesarenotdependentontheoriginofproteins,theusedpromoters,thehydrophobicityoftargetproteins...第7页,共65页,2023年,2月20日,星期四SomeE.coliexpressionhostconsiderations第8页,共65页,2023年,2月20日,星期四YeastExpression第9页,共65页,2023年,2月20日,星期四YeastExpressionYeastisaeukaryoticorganismthatcanbegrowntoveryhighdensities,whichmakesthemespeciallyusefulfortheproductionofisotopelabeledproteinforNMR.UsefulstrainsincludeSaccharomycescerevisiaeandthemethylotrophicyeastPichiapastoris.Theyeaststrainshavebeengeneticallywellcharacterizedandareknowntoperformmanyposttranslationalmodifications.Yeastcangrowquicklyindefinedmedium,areeasierandlessexpensivetoworkwiththaninsectormammaliancells,andareeasilyadaptedtofermentation.Yeastexpressionisideallysuitedforlarge-scaleproductionofrecombinanteukaryoticproteins.Themajoradvantagesofyeastexpression:highyieldandproductivity,highcelldensities,andsuperiorexpression.第10页,共65页,2023年,2月20日,星期四ControllableprocessThegrowthmediumthatfeedsyeastiscompletelydefined.Itconsistsofasimple,inexpensiveformulation.Thecarbonsourceisfedtothefermentorataratedesignedtoachievemaximumcelldensitywhilemaintainingoptimalproductionofforeignprotein.Thisprocessminimizesanytoxiceffectstheforeignproteinmighthaveontheyeast.ProductprocessingsimilartomammaliancellsTheyeastexpressionsystemproducesmammalian-likeproteins.Forexample,theexpressionofHepatitisBsurfaceantigen(HBsAg)inyeastleadstoproductionofparticlesthatareimmunoreactivewithanti-HBsAgantibodies.TheseparticlesaresimilartoDaneparticlesisolatedfromtheseraofhumancarriers.

StableproductionstrainsExpressionofforeigngenesisachievedbyintegrationofforeignDNAintothechromosomalDNAofhostgenome.TheintegratedDNAisstableformanygenerations;allcellscanproducetheprotein.Incontrast,plasmid-basedsystemsrequireselectivepressureonplasmidstomaintaintheforeignDNA.Cellsthatlosetheplasmidcannotproducethedesiredforeignprotein.DurabilityTheYeastExpressionSystemrequiresnospecialhandling.Itwasdevelopedtowithstandtheadverseconditionsoflargescale,continuousfermentors.Thisfeaturemakesyeastabletosurviveunexpecteddisruptionsinthefermentationprocess

LowerproteinproductioncostHighper-cellexpressionlevelscombinedwithhighcell-densitygrowthofyeasttranslatesintogreaterquantitiesofrecombinantproteinperfermentorvolume.Thisreducesproductioncostsbyincreasingtheamountofproductperfermentationrun.Proteinpurificationisanothercost-savingarea.Theyeastsystemcansecreteproteinintothemedium,sothebroththatenterspurificationcontainsahigherconcentrationofthedesiredprotein.Pureproteinisrecoveredwithhigheryieldandlowercost.YeastExpression(Cont.)第11页,共65页,2023年,2月20日,星期四ExpressioninYeastAutonomousreplicatingvectors->shuttlevectors第12页,共65页,2023年,2月20日,星期四ExpressioninSaccharomycescerevisiae

Autonomousreplicatingsystems第13页,共65页,2023年,2月20日,星期四ExpressioninSaccharomycescerevisiae

IntegrativesystemsProbabilityforintegrationhigherwithlinearfragments!第14页,共65页,2023年,2月20日,星期四ExpressioninSaccharomycescerevisiae第15页,共65页,2023年,2月20日,星期四ExpressioninS.cerevisiaeVs.PichiapastorisProblemswithproductioninS.cerevisiae:Forsomeproteinsproductionlevelislow.Hyperglycosylation(morethan100mannoseresiduesinN-glycosylation).Sometimessecretionisnotgood->proteinstackincells(periplasma).S.cerevisiaeproduceshighamountofEtOH->toxicforthecells->affectslevelofproduction.AdvantagesofproductioninPichiapastoris:

Highlyefficientpromoter,tightlyregulated(alcoholoxidase->AOX,inducedbyMeOH).ProducesnoEtOH->veryhighcelldensity->secretionveryefficient.Secretesveryfewproteins->simplificationofpurificationofsecretedproteins.第16页,共65页,2023年,2月20日,星期四InsectExpressionBaculovirusorDrosophila第17页,共65页,2023年,2月20日,星期四BaculovirusBaculovirusarepresentininvertebratesprimarilyinsectspecies.Theyarenotinfectiousforvertebrates&plants.Genomeiscovalentlyclosedcirculardoublestrandedof134kbp,duetoitssmallitcanaccommodatelargefragmentsofforeignDNA.TheyaredividedintotwogroupsonthebasisoftheirstructureasNucleopolyhedroviruses(NPV)andGranuloviruses.TheseNPVaremainlyusedasexpressionvectors,i.e.AutographacalifornicaNPV(AcMNPV)isolatedfromthelarvaofthealfalfalooper.BaculovirusexpressionsystembasedupontheabilitytopropagateAcMNPVininsectcells.Usesmanyoftheproteinmodification,processingandtransportsystemspresentinhighereukaryoticcells.Virusthatcanbepropagatedtohightitersadaptedforgrowthinsuspensionculturesobtainlargeamountsofrecombinantproteinwithrelativeease.Baculovirusarenoninfectioustovertebratesandtheirpromotersareinactiveinmammaliancells.第18页,共65页,2023年,2月20日,星期四AdvantagesofworkingwithBaclosystemHighexpressionlevelsusingthepolyhedrinorp10promoterSupportspost-translationmodificationsBEVSenablessimultaneousexpressionofmultiplegenesExpressedproteinsdonothavesizelimitationsCapableofproducingcytotoxicproteinsLeukemiainworkingwithBEVSBaculovirussystemworksonlyininvertebratessotheexpressedvertebrateproteinsaredifferentinposttranslationmodificationswithhighmannosetypeglycosylation.Ithaslimitedcapacitytoproperlyprocessedinactiveprecursorproteinsduetotheabsenceofpro-proteinconvertasesLimitedproteinyieldduetoaccumulationofinsolubleproteinwithinthecells第19页,共65页,2023年,2月20日,星期四Insects&InsectcellsBaculovirusinfectslepidopteran(butterflies&moths)insectsandinsectcelllines.Commonlyusedcelllinesaresf9&sf21derivedfromthepupalovariantissueofthefallarmywormspodopterafrugiperdaandhighfivederivedfromtheovariancellsofthecabbagelooper.第20页,共65页,2023年,2月20日,星期四TypesofInsectcelllinescellsDoublingtimeCellappearanceMediumOriginTypeofcultureSf972hrsSpherical,granular,regularinsize,firmattachmenttosurfaceTNM-FHIPLBSF-21celllinesofthefallarmywormspodopterafrugiperdaGrowwellasmonolayerandsuspensionSf2124hrsSpherical,granular,differentinsize,firmattachmenttosurfaceTNM-FHIPLBSF-21celllinesofthefallarmywormspodopterafrugiperdaGrowwellasmonolayerandsuspensionHigh-five18hrsSpherical,granular,regularinsize,looseattachmenttosurfaceExpressfiveSFMOvariancellsofcabbagelooperGrowwellasmonolayer,alsoassuspension第21页,共65页,2023年,2月20日,星期四StepsinrecombinantbaculovirusproductionClonethegeneofinterestinpfastBacdonorplasmid.ExpressioncassetteinpfastBacisflankedbyleftandrightarmsofTn7andalsoanSV40polyadenylationsignaltoformaminiTn7.ClonedpfastBacistransformedinE.colihoststrain(DH10Bac)whichcontainsabaculovirusshuttlevectorbacmidhavingamini-attTn7targetsite.Helperplasmidwhichallowstotransposethegeneofinterestfrompfasttobacmid(shuttlevector).Transpositionoccursbetweenthemini-attTn7targetsitetogeneratearecombinantbacmid.Thisrecombinantbacmidcannowbeusedtotransfectinsectcelllines.第22页,共65页,2023年,2月20日,星期四128bp145bpMiniattTn7M13forwardM13reverseTn7RGOITn7LBacmidDNATransposedpfastBacGeneofInterestTn7RPpHTn7LpfastBacwithinsertFigures:第23页,共65页,2023年,2月20日,星期四Alternative:Homogeneousrecombination第24页,共65页,2023年,2月20日,星期四InsectMediumGrace’sInsectmedium-unsupplementedbutcontainsL-glutamineGrace’sInsectmediumsupplemented-containsadditionalTCyeastolate&LactalbuminhydrolysateTrichoplusianiMediumformulationhink(TNM-FH)-contains10%FBSSerum-freemediumSFM-900andExpress-Five第25页,共65页,2023年,2月20日,星期四RequirementsforpropercellcultureTemperature-Optimalrangeis27-28ºCpH-Optimalrangeis6.1to6.4Aeration-Requirespassive02diffusionforoptimalgrowth&recombinantproteinexpressionOsmolality-Optimumis345-380mOsm/kgFBS-Workingwithsuspensioncultureitisadvisabletouse(10-20%FBS)togaveprotectionfromcellularshearforces第26页,共65页,2023年,2月20日,星期四MethodsofsubculturingadherentcellsThreemethodstodislodgemonolayersinadherentcellculture-Sloughing-Trypsinization-TappingthelayeruntilmonolayerloosensTypesofcellculturingMonolayercultureSuspensionculture第27页,共65页,2023年,2月20日,星期四ProcedureofmonolayersubcultureMonolayershouldreachtoconfluencyin2-4days.SerumsupplementedculturesdonotadheretosurfacetightlywhereasserumfreeattachverytightlytosubstratesAspiratemedium&floatingcellsfromaconfluentmonolayer&discardthem.Add4mlofRTcompletegrowthmediumtoeach25cm2flask(12mltoa75cm2flask)ResuspendcellsbypipettingthemediumacrossthemonolayerwithaPasteurpipette.(Enzymaticdissociationisnotrecommended)ObservecellmonolayerusinganinvertedmicroscopetoensureadequatecelldetachmentPerformviablecellscountonharvestedcells.Inoculatecellsat2x105viablecells/mlintorespectiveculturevessels.Inoculatecultureskeptat25-28ºCwithloosecapstoallowgaseousexchangeOnday4post-planting,aspiratethespentmediumfromonesideofthemonolayer&subculturetheflaskWithslowergrowingcelllines,itmaybenecessarytofeedtheflasksonday3-4postplantingSubculturetheflaskswhenthemonolayerreaches80-100%confluency,approx2-3dayspostplanting第28页,共65页,2023年,2月20日,星期四WorkingwithsuspensioncultureInsectcellsarenotgenerallyanchoragedependent&canbewelladaptedtosuspensionculturePriortoestablishaspinnerculture,cellsaremaintainedfirstlyashealthyadherentcells.Celldensityreachesto2-2.5x106cells/mltheyshouldbedilutedtonolessthan7x105cells/mlUseaspinnerflaskwithaverticalimpellerCulturevolumeshouldnotexceedhalfofthevolumeoftheflaskUseofsurfactanttodecreaseshearinge.g.PluronicF-68Notnecessarytochangemediumregularly.Subculturingrequirestheremovalofcellsuspension&theadditionofmediumImpellershouldberotatingregularlyImpellershouldbesubmerged1cmormoretoensureadequateaerationCellviabilityof95%isrequiredMinimumdensityof1x106cells/mlisrequiredKeeprecordofthepassagenumber.After30passageormore(2-3months),cellsdoublingtimeincreasedandalsoloosetheirviabilityandinfectivity.Keepacelllog,todosooneshouldhaveaknowledgeoffollowing:dateofinitiationofculture,lotnumber;dateofpassage&passagenumber;density&viabilityatpassage;commentoncellappearance;medium&itslotnumber第29页,共65页,2023年,2月20日,星期四CryopreseravtionofcellsFreezingcellsshouldbe90%viableand80-90%confluent.Freezingmediumshouldhave60%Grace’sinsectmediumsupplementedwith30%FBS&10%DMSO.Countcellsusinghaemocytometer.Placedcryovialsonice&labelthem.Centrifugecellsat400-600gfor10mtsatRT.Removethesupernatant.Resuspendthecellstothegivendensityinthefreezingmedium.Transfer1mlofthecellsuspensiontosterilecryovials.Placeat-20ºCfor1hrthentransferto-80ºCfor24-48hrs&thenfinallystoreatLiquidnitrogen.第30页,共65页,2023年,2月20日,星期四DoandDon'tsCheckcellsdailyuntilaconfluentmonolayerisformed.Passagecellsatconfluencyonly,ascellswillbeeasytodislodge&showsbetterviability.Donotovergrowcells,itresultsindecreasedviability.Donotsplitscellstoofar.Densitieslowerthan20%confluencyinhibitgrowth.Passagethecellsonlyinlogphase,logphasegrowthcanbemaintainedbysplittingcellsin1:5dilution.第31页,共65页,2023年,2月20日,星期四TheexpressionofproteincomplexesImprovethesolubilityandstabilityofproteins,especiallyforbigproteinproducts.Thetagsused.Themutualinfluencesofco-expressedproteins.Example:Bcl-xl/Bim/LC8.第32页,共65页,2023年,2月20日,星期四ODVolumeOD280Volume140KD68KD¬21¬31Bcl-xLBimLC8¬14ABPurificationofBcl-xl/Bim/LC8complex第33页,共65页,2023年,2月20日,星期四DrosophilaTheDrosophilaExpressionSystemutilizesacelllinederivedfromDrosophilamelanogaster,Schneider2(S2)cells,andasimpleplasmidvectorwithmetallothioneinpromotor(coppersulfateinducible)fortheexpressionofheterologousproteins.S2cellsareeasilymaintainedinlooselyadherentorsuspensioncultureatroomtemperatureanddonotrequireCO2.第34页,共65页,2023年,2月20日,星期四MammalianExpression第35页,共65页,2023年,2月20日,星期四MammalianExpressionTheproductionofproteinsinmammaliancellsisanimportanttoolinnumerousscientificandcommercialareas.Theproteinsexpressedinandpurifiedfrommammaliancellsystemareroutinelyneededforlifescienceresearchanddevelopment.

Inthefieldofbiomedicine,proteinsforhumantherapy,vaccinationordiagnosticapplicationsaretypicallyproducedinmammaliancells.

Genecloning,proteinengineering,biochemicalandbiophysicalcharacterizationofproteinsalsorequiretheuseofgeneexpressioninmammaliancells.

Otherapplicationsinwidespreaduseinvolvescreeningoflibrariesofchemicalcompoundsindrugdiscovery,andthedevelopmentofcell-basedbiosensors.第36页,共65页,2023年,2月20日,星期四MammalianExpression(cont.)Themajoradvantagesofmammalianexpression:ProperproteinfoldingandfunctionHighsuccessrateNaturalproteinconfigurationBestPost-translationalmodification(PTM)Developedcelllines:shortterm(transient)expression->autonomousreplicatingsystems->viralorigins(SV40)-Africangreenmonkeykidney(COS)-babyhamsterkidney(BHK)-humanembryonickidney(HEK-239)longterm(stable)expression->integrationintochromosome->viralorigins-chinesehamsterovary(CHO)第37页,共65页,2023年,2月20日,星期四NucleusERRERGolgiONLYCORRECTLYFOLDEDPROTEINSARESECRETEDPROTEINEXPRESSIONINMAMMALIANCELLSS-Sformation(ER)Glycosylation(ER+Golgi)Qualitycontrol(ER)第38页,共65页,2023年,2月20日,星期四COMMONLYUSEDMAMMALIANCELLSHEK293:HumanembryonickidneycellsCHO:ChineseHamsterOvarycellsCOS:Simianfibroblasts第39页,共65页,2023年,2月20日,星期四

TISSUECULTUREMostmammaliancellsareadherentCulturedinplatesorflasksGrowinmonolayeronspeciallytreatedsurfacesMediumsupplementedwith5-10%FetalCalfSerumLaminarflowcabinetCO2incubator第40页,共65页,2023年,2月20日,星期四

EXPRESSIONVECTORStrongpromoter(CMV)AntibioticresistancegeneforselectionofstablecelllineAntibioticresistancegeneforE.coliselection

NOTALWAYSINCLUDEDBUTESSENTIALLeadersequence第41页,共65页,2023年,2月20日,星期四

TRANSFECTIONOFMAMMALIANCELLSElectroporationCa-phosphateLiposomebasedtransfectionreagents

TYPESOFTRANSFECTIONTransientStableEpisomal第42页,共65页,2023年,2月20日,星期四TRANSIENTTRANSFECTIONGenetoproteinindaysTestingexpressionFunctionalstudiesLowyieldUsedinhigh-throughputstructuralstudies(293cells)第43页,共65页,2023年,2月20日,星期四STABLETRANSFECTIONGenetoproteinin≥2monthsComplexprocessGeneofinterestintegratesintogenomeofhostcellHighyields(from1to5mg/landhigher)Stockofcellsexpressingdesiredrecombinantprotein第44页,共65页,2023年,2月20日,星期四STABLETRANSFECTIONSelectionpressureScreeningclonesforexpressionCloningofpositiveclonesScreeningofsingleclonesforexpressionTransfectionScalingup第45页,共65页,2023年,2月20日,星期四

EPISOMALTRANSFECTIONGenetolargescaleproteinproductionin~4weeksStraightforwardprocessHEKEBNAcells(293stablytransfectedwithEBNA-1gene)EBNA-1drivenepisomalreplicationof

Ori-PcontainingvectorsVeryhighyields(5to20mg/landhigher)第46页,共65页,2023年,2月20日,星期四EASY2STEPSPROTEINPURIFICATIONAFFINITYCHROMATOGRAPHYGELFILTRATION0500Absorptionat280nm(mAU)1000150020002500500mMImidazole-45kDaElutionvolume(ml)Vo10152025050010001500Absorptionat280nm(mAU)2000-45kDa第47页,共65页,2023年,2月20日,星期四

GLYCOSYLATIONMammaliansugarchainshavehighlycomplexstructuresGoodforfunctionalstudiesBigproblemforproteincrystallizationSOLUTIONSMutagenesisofglycosylationsitesEnzymaticdeglycosylationEngineeredcelllines(CHOLecstrains)ChemicalinhibitorsofglycosylationpathwayInsectcells(simplersugars)第48页,共65页,2023年,2月20日,星期四EXTRACELLULARPROTEINSLargeextracellulardomainsModularmultidomainorganisationPosttranslationalmodificationsDisulfidebridgesGlycosylation

INTEGRINαVβ39domains28disulfidebridges6N-linkedglycosylationsites第49页,共65页,2023年,2月20日,星期四Summary:Competitivenessofdifferentexpressionsystems第50页,共65页,2023年,2月20日,星期四在蛋白质的鉴定和分析中常用的技术方法第51页,共65页,2023年,2月20日,星期四盐析法等电点沉淀法离子交换纤维素色谱葡聚糖凝胶色谱亲和色谱蛋白质的纯化方法第52页,共65页,2023年,2月20日,星期四材料的预处理及细胞破碎机械破碎法渗透破碎法反复冻融法超声波法酶法第53页,共65页,2023年,2月20日,星期四蛋白质粗制品的获得等电点沉淀法盐析法有机溶剂沉淀法第54页,共65页,2023年,2月20日,星期四蛋白质的纯化方法凝胶过滤法(分子排阻色谱,分子筛色谱):根据蛋白质分子量大小离子交换纤维素柱色谱法:根据蛋白质所带电荷不同,酸碱性质亲和色谱:抗体,各种标签,根据蛋白质对特定分子物质具有专一性结合的原理高效(压)液相色谱(HPLC):根据蛋白质在有机溶剂里迁移率的不同疏水色谱:根据蛋白质的疏水性质第55页,共65页,2023年,2月20日,星期四凝胶过滤法:蛋白质分子量的对数和洗脱体积之间成线性关系(反比例)SDS-聚丙烯酰胺凝胶电泳法:SDS与蛋白质结合使其带负电荷,电泳结果只与分子量有关,与所带电荷无关,分子量的对数与迁移率之间有线性关系(反比例)超速离心法:利用蛋白质密度不同,经超速离心后,分布于不同的液层而分离,蛋白质的分子量与其沉降系数S成正比分子量测定的方法第56页,共65页,2023年,2月20日,星期四蛋白的纯度和均一性鉴别标准蛋白质在SDS呈现一条分离带等电聚焦电泳呈现一条分离带在超速离心场中,以单一的沉降速度运动在色谱分析中呈现一个洗脱峰经纯化后的蛋白质不失活性第57页,共65页,2023年,2月20日,星期四紫外吸收法:A280吸收值福林酚法(Folin法):又称Lowry法,蛋白质与福林试

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