高效蛋白表達(dá)系統(tǒng)第一頁(yè)，共六十五頁(yè)，編輯于2023年，星期六ProteinExpressionProteinexpressionisasubcomponentofgeneexpression.ItconsistsofthestagesafterDNAhasbeentranslatedintoaminoacidchains,whichareultimatelyfoldedintoproteins.Initssimplestform,aproteinexpressionsysteminvolvesatemplate,amechanismoftranscription/translationsuchasacellorcellextract,andtherawmaterialsrequiredtobuildproteins.Proteinexpressioncanbedoneinprokaryotesiftheproteindoesn’tneedtobepost-translationallymodified.Ifperformingstructure-functionstudies,expressinginaeukaryoticsystemisimportantbecausethesecellshavethepropercellularmachinerytodecoratetheproteininquestionwiththecorrectpost-translationalmodifications.Thetypesofeukaryoticcellstypicallyusedinproteinexpressionincludeyeast,insect,andmammalian.第二頁(yè)，共六十五頁(yè)，編輯于2023年，星期六ChoiceoftheexpressionsystemCell-freeBacteriaYeastInsectMammalianEasyofuseCostofmediaandEquipmentPos-translationalModifications(Probabilityofproteinfunction)TimeRequirement第三頁(yè)，共六十五頁(yè)，編輯于2023年，星期六ProkaryoticExpression第四頁(yè)，共六十五頁(yè)，編輯于2023年，星期六ProteinexpressioninprokaryoticsystemBacterialexpressionvectorshavesomedistinctfeatures:

Induciblepromotersystems;Proteinfusionsincludingfusedtags;Easymanipulation;第五頁(yè)，共六十五頁(yè)，編輯于2023年，星期六SomeproblemsofproductioninE.coli

第六頁(yè)，共六十五頁(yè)，編輯于2023年，星期六Inclusionbodies(mostcommoncase)

Inclusionbodiesareformed

throughtheaccumulationoffoldingintermediates

ratherthanfromthenativeorunfoldedproteins.

Itisnotpossibletopredictwhichproteinswillbeproducedasinclusionbodies.

Productionofinclusionbodiesarenotdependentontheoriginofproteins,theusedpromoters,thehydrophobicityoftargetproteins...第七頁(yè)，共六十五頁(yè)，編輯于2023年，星期六SomeE.coliexpressionhostconsiderations第八頁(yè)，共六十五頁(yè)，編輯于2023年，星期六YeastExpression第九頁(yè)，共六十五頁(yè)，編輯于2023年，星期六YeastExpressionYeastisaeukaryoticorganismthatcanbegrowntoveryhighdensities,whichmakesthemespeciallyusefulfortheproductionofisotopelabeledproteinforNMR.UsefulstrainsincludeSaccharomycescerevisiaeandthemethylotrophicyeastPichiapastoris.Theyeaststrainshavebeengeneticallywellcharacterizedandareknowntoperformmanyposttranslationalmodifications.Yeastcangrowquicklyindefinedmedium,areeasierandlessexpensivetoworkwiththaninsectormammaliancells,andareeasilyadaptedtofermentation.Yeastexpressionisideallysuitedforlarge-scaleproductionofrecombinanteukaryoticproteins.Themajoradvantagesofyeastexpression:highyieldandproductivity,highcelldensities,andsuperiorexpression.第十頁(yè)，共六十五頁(yè)，編輯于2023年，星期六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.)第十一頁(yè)，共六十五頁(yè)，編輯于2023年，星期六ExpressioninYeastAutonomousreplicatingvectors->shuttlevectors第十二頁(yè)，共六十五頁(yè)，編輯于2023年，星期六ExpressioninSaccharomycescerevisiae

Autonomousreplicatingsystems第十三頁(yè)，共六十五頁(yè)，編輯于2023年，星期六ExpressioninSaccharomycescerevisiae

IntegrativesystemsProbabilityforintegrationhigherwithlinearfragments!第十四頁(yè)，共六十五頁(yè)，編輯于2023年，星期六ExpressioninSaccharomycescerevisiae第十五頁(yè)，共六十五頁(yè)，編輯于2023年，星期六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.第十六頁(yè)，共六十五頁(yè)，編輯于2023年，星期六InsectExpressionBaculovirusorDrosophila第十七頁(yè)，共六十五頁(yè)，編輯于2023年，星期六BaculovirusBaculovirusarepresentininvertebratesprimarilyinsectspecies.Theyarenotinfectiousforvertebrates&plants.Genomeiscovalentlyclosedcirculardoublestrandedof134kbp,duetoitssmallitcanaccommodatelargefragmentsofforeignDNA.TheyaredividedintotwogroupsonthebasisoftheirstructureasNucleopolyhedroviruses(NPV)andGranuloviruses.TheseNPVaremainlyusedasexpressionvectors,i.e.AutographacalifornicaNPV(AcMNPV)isolatedfromthelarvaofthealfalfalooper.BaculovirusexpressionsystembasedupontheabilitytopropagateAcMNPVininsectcells.Usesmanyoftheproteinmodification,processingandtransportsystemspresentinhighereukaryoticcells.Virusthatcanbepropagatedtohightitersadaptedforgrowthinsuspensionculturesobtainlargeamountsofrecombinantproteinwithrelativeease.Baculovirusarenoninfectioustovertebratesandtheirpromotersareinactiveinmammaliancells.第十八頁(yè)，共六十五頁(yè)，編輯于2023年，星期六AdvantagesofworkingwithBaclosystemHighexpressionlevelsusingthepolyhedrinorp10promoterSupportspost-translationmodificationsBEVSenablessimultaneousexpressionofmultiplegenesExpressedproteinsdonothavesizelimitationsCapableofproducingcytotoxicproteinsLeukemiainworkingwithBEVSBaculovirussystemworksonlyininvertebratessotheexpressedvertebrateproteinsaredifferentinposttranslationmodificationswithhighmannosetypeglycosylation.Ithaslimitedcapacitytoproperlyprocessedinactiveprecursorproteinsduetotheabsenceofpro-proteinconvertasesLimitedproteinyieldduetoaccumulationofinsolubleproteinwithinthecells第十九頁(yè)，共六十五頁(yè)，編輯于2023年，星期六Insects&InsectcellsBaculovirusinfectslepidopteran(butterflies&moths)insectsandinsectcelllines.Commonlyusedcelllinesaresf9&sf21derivedfromthepupalovariantissueofthefallarmywormspodopterafrugiperdaandhighfivederivedfromtheovariancellsofthecabbagelooper.第二十頁(yè)，共六十五頁(yè)，編輯于2023年，星期六TypesofInsectcelllinescellsDoublingtimeCellappearanceMediumOriginTypeofcultureSf972hrsSpherical,granular,regularinsize,firmattachmenttosurfaceTNM-FHIPLBSF-21celllinesofthefallarmywormspodopterafrugiperdaGrowwellasmonolayerandsuspensionSf2124hrsSpherical,granular,differentinsize,firmattachmenttosurfaceTNM-FHIPLBSF-21celllinesofthefallarmywormspodopterafrugiperdaGrowwellasmonolayerandsuspensionHigh-five18hrsSpherical,granular,regularinsize,looseattachmenttosurfaceExpressfiveSFMOvariancellsofcabbagelooperGrowwellasmonolayer,alsoassuspension第二十一頁(yè)，共六十五頁(yè)，編輯于2023年，星期六StepsinrecombinantbaculovirusproductionClonethegeneofinterestinpfastBacdonorplasmid.ExpressioncassetteinpfastBacisflankedbyleftandrightarmsofTn7andalsoanSV40polyadenylationsignaltoformaminiTn7.ClonedpfastBacistransformedinE.colihoststrain(DH10Bac)whichcontainsabaculovirusshuttlevectorbacmidhavingamini-attTn7targetsite.Helperplasmidwhichallowstotransposethegeneofinterestfrompfasttobacmid(shuttlevector).Transpositionoccursbetweenthemini-attTn7targetsitetogeneratearecombinantbacmid.Thisrecombinantbacmidcannowbeusedtotransfectinsectcelllines.第二十二頁(yè)，共六十五頁(yè)，編輯于2023年，星期六128bp145bpMiniattTn7M13forwardM13reverseTn7RGOITn7LBacmidDNATransposedpfastBacGeneofInterestTn7RPpHTn7LpfastBacwithinsertFigures:第二十三頁(yè)，共六十五頁(yè)，編輯于2023年，星期六Alternative:Homogeneousrecombination第二十四頁(yè)，共六十五頁(yè)，編輯于2023年，星期六InsectMediumGrace’sInsectmedium-unsupplementedbutcontainsL-glutamineGrace’sInsectmediumsupplemented-containsadditionalTCyeastolate&LactalbuminhydrolysateTrichoplusianiMediumformulationhink(TNM-FH)-contains10%FBSSerum-freemediumSFM-900andExpress-Five第二十五頁(yè)，共六十五頁(yè)，編輯于2023年，星期六RequirementsforpropercellcultureTemperature-Optimalrangeis27-28oCpH-Optimalrangeis6.1to6.4Aeration-Requirespassive02diffusionforoptimalgrowth&recombinantproteinexpressionOsmolality-Optimumis345-380mOsm/kgFBS-Workingwithsuspensioncultureitisadvisabletouse(10-20%FBS)togaveprotectionfromcellularshearforces第二十六頁(yè)，共六十五頁(yè)，編輯于2023年，星期六MethodsofsubculturingadherentcellsThreemethodstodislodgemonolayersinadherentcellculture-Sloughing-Trypsinization-TappingthelayeruntilmonolayerloosensTypesofcellculturingMonolayercultureSuspensionculture第二十七頁(yè)，共六十五頁(yè)，編輯于2023年，星期六ProcedureofmonolayersubcultureMonolayershouldreachtoconfluencyin2-4days.SerumsupplementedculturesdonotadheretosurfacetightlywhereasserumfreeattachverytightlytosubstratesAspiratemedium&floatingcellsfromaconfluentmonolayer&discardthem.Add4mlofRTcompletegrowthmediumtoeach25cm2flask(12mltoa75cm2flask)ResuspendcellsbypipettingthemediumacrossthemonolayerwithaPasteurpipette.(Enzymaticdissociationisnotrecommended)ObservecellmonolayerusinganinvertedmicroscopetoensureadequatecelldetachmentPerformviablecellscountonharvestedcells.Inoculatecellsat2x105viablecells/mlintorespectiveculturevessels.Inoculatecultureskeptat25-28oCwithloosecapstoallowgaseousexchangeOnday4post-planting,aspiratethespentmediumfromonesideofthemonolayer&subculturetheflaskWithslowergrowingcelllines,itmaybenecessarytofeedtheflasksonday3-4postplantingSubculturetheflaskswhenthemonolayerreaches80-100%confluency,approx2-3dayspostplanting第二十八頁(yè)，共六十五頁(yè)，編輯于2023年，星期六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第二十九頁(yè)，共六十五頁(yè)，編輯于2023年，星期六CryopreseravtionofcellsFreezingcellsshouldbe90%viableand80-90%confluent.Freezingmediumshouldhave60%Grace’sinsectmediumsupplementedwith30%FBS&10%DMSO.Countcellsusinghaemocytometer.Placedcryovialsonice&labelthem.Centrifugecellsat400-600gfor10mtsatRT.Removethesupernatant.Resuspendthecellstothegivendensityinthefreezingmedium.Transfer1mlofthecellsuspensiontosterilecryovials.Placeat-20oCfor1hrthentransferto-80oCfor24-48hrs&thenfinallystoreatLiquidnitrogen.第三十頁(yè)，共六十五頁(yè)，編輯于2023年，星期六DoandDon'tsCheckcellsdailyuntilaconfluentmonolayerisformed.Passagecellsatconfluencyonly,ascellswillbeeasytodislodge&showsbetterviability.Donotovergrowcells,itresultsindecreasedviability.Donotsplitscellstoofar.Densitieslowerthan20%confluencyinhibitgrowth.Passagethecellsonlyinlogphase,logphasegrowthcanbemaintainedbysplittingcellsin1:5dilution.第三十一頁(yè)，共六十五頁(yè)，編輯于2023年，星期六TheexpressionofproteincomplexesImprovethesolubilityandstabilityofproteins,especiallyforbigproteinproducts.Thetagsused.Themutualinfluencesofco-expressedproteins.Example:Bcl-xl/Bim/LC8.第三十二頁(yè)，共六十五頁(yè)，編輯于2023年，星期六ODVolumeOD280Volume140KD68KD?21?31Bcl-xLBimLC8?14ABPurificationofBcl-xl/Bim/LC8complex第三十三頁(yè)，共六十五頁(yè)，編輯于2023年，星期六DrosophilaTheDrosophilaExpressionSystemutilizesacelllinederivedfromDrosophilamelanogaster,Schneider2(S2)cells,andasimpleplasmidvectorwithmetallothioneinpromotor(coppersulfateinducible)fortheexpressionofheterologousproteins.S2cellsareeasilymaintainedinlooselyadherentorsuspensioncultureatroomtemperatureanddonotrequireCO2.第三十四頁(yè)，共六十五頁(yè)，編輯于2023年，星期六MammalianExpression第三十五頁(yè)，共六十五頁(yè)，編輯于2023年，星期六MammalianExpressionTheproductionofproteinsinmammaliancellsisanimportanttoolinnumerousscientificandcommercialareas.Theproteinsexpressedinandpurifiedfrommammaliancellsystemareroutinelyneededforlifescienceresearchanddevelopment.

Inthefieldofbiomedicine,proteinsforhumantherapy,vaccinationordiagnosticapplicationsaretypicallyproducedinmammaliancells.

Genecloning,proteinengineering,biochemicalandbiophysicalcharacterizationofproteinsalsorequiretheuseofgeneexpressioninmammaliancells.

Otherapplicationsinwidespreaduseinvolvescreeningoflibrariesofchemicalcompoundsindrugdiscovery,andthedevelopmentofcell-basedbiosensors.第三十六頁(yè)，共六十五頁(yè)，編輯于2023年，星期六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)第三十七頁(yè)，共六十五頁(yè)，編輯于2023年，星期六NucleusERRERGolgiONLYCORRECTLYFOLDEDPROTEINSARESECRETEDPROTEINEXPRESSIONINMAMMALIANCELLSS-Sformation(ER)Glycosylation(ER+Golgi)Qualitycontrol(ER)第三十八頁(yè)，共六十五頁(yè)，編輯于2023年，星期六COMMONLYUSEDMAMMALIANCELLSHEK293:HumanembryonickidneycellsCHO:ChineseHamsterOvarycellsCOS:Simianfibroblasts第三十九頁(yè)，共六十五頁(yè)，編輯于2023年，星期六

TISSUECULTUREMostmammaliancellsareadherentCulturedinplatesorflasksGrowinmonolayeronspeciallytreatedsurfacesMediumsupplementedwith5-10%FetalCalfSerumLaminarflowcabinetCO2incubator第四十頁(yè)，共六十五頁(yè)，編輯于2023年，星期六

EXPRESSIONVECTORStrongpromoter(CMV)AntibioticresistancegeneforselectionofstablecelllineAntibioticresistancegeneforE.coliselection

NOTALWAYSINCLUDEDBUTESSENTIALLeadersequence第四十一頁(yè)，共六十五頁(yè)，編輯于2023年，星期六

TRANSFECTIONOFMAMMALIANCELLSElectroporationCa-phosphateLiposomebasedtransfectionreagents

TYPESOFTRANSFECTIONTransientStableEpisomal第四十二頁(yè)，共六十五頁(yè)，編輯于2023年，星期六TRANSIENTTRANSFECTIONGenetoproteinindaysTestingexpressionFunctionalstudiesLowyieldUsedinhigh-throughputstructuralstudies(293cells)第四十三頁(yè)，共六十五頁(yè)，編輯于2023年，星期六STABLETRANSFECTIONGenetoproteinin≥2monthsComplexprocessGeneofinterestintegratesintogenomeofhostcellHighyields(from1to5mg/landhigher)Stockofcellsexpressingdesiredrecombinantprotein第四十四頁(yè)，共六十五頁(yè)，編輯于2023年，星期六STABLETRANSFECTIONSelectionpressureScreeningclonesforexpressionCloningofpositiveclonesScreeningofsingleclonesforexpressionTransfectionScalingup第四十五頁(yè)，共六十五頁(yè)，編輯于2023年，星期六

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

Ori-PcontainingvectorsVeryhighyields(5to20mg/landhigher)第四十六頁(yè)，共六十五頁(yè)，編輯于2023年，星期六EASY2STEPSPROTEINPURIFICATIONAFFINITYCHROMATOGRAPHYGELFILTRATION0500Absorptionat280nm(mAU)1000150020002500500mMImidazole-45kDaElutionvolume(ml)Vo10152025050010001500Absorptionat280nm(mAU)2000-45kDa第四十七頁(yè)，共六十五頁(yè)，編輯于2023年，星期六

GLYCOSYLATIONMammaliansugarchainshavehighlycomplexstructuresGoodforfunctionalstudiesBigproblemforproteincrystallizationSOLUTIONSMutagenesisofglycosylationsitesEnzymaticdeglycosylationEngineeredcelllines(CHOLecstrains)ChemicalinhibitorsofglycosylationpathwayInsectcells(simplersugars)第四十八頁(yè)，共六十五頁(yè)，編輯于2023年，星期六EXTRACELLULARPROTEINSLargeextracellulardomainsModularmultidomainorganisationPosttranslationalmodificationsDisulfidebridgesGlycosylation

INTEGRINαVβ39domains28disulfidebridges6N-linkedglycosylationsites第四十九頁(yè)，共六十五頁(yè)，編輯于2023年，星期六Summary:Competitivenessofdifferentexpressionsystems第五十頁(yè)，共六十五頁(yè)，編輯于2023年，星期六在蛋白質(zhì)的鑒定和分析中常用的技術(shù)方法第五十一頁(yè)，共六十五頁(yè)，編輯于2023年，星期六鹽析法等電點(diǎn)沉淀法離子交換纖維素色譜葡聚糖凝膠色譜親和色譜蛋白質(zhì)的純化方法第五十二頁(yè)，共六十五頁(yè)，編輯于2023年，星期六材料的預(yù)處理及細(xì)胞破碎機(jī)械破碎法滲透破碎法反復(fù)凍融法超聲波法酶法第五十三頁(yè)，共六十五頁(yè)，編輯于2023年，星期六蛋白質(zhì)粗制品的獲得等電點(diǎn)沉淀法鹽析法有機(jī)溶劑沉淀法第五十四頁(yè)，共六十五頁(yè)，編輯于2023年，星期六蛋白質(zhì)的純化方法凝膠過(guò)濾法（分子排阻色譜，分子篩色譜）：根據(jù)蛋白質(zhì)分子量大小離子交換纖維素柱色譜法：根據(jù)蛋白質(zhì)所帶電荷不同，酸堿性質(zhì)親和色譜：抗體，各種標(biāo)簽，根據(jù)蛋白質(zhì)對(duì)特定分子物質(zhì)具有專(zhuān)一性結(jié)合的原理高效（壓）液相色譜（HPLC）：根據(jù)蛋白質(zhì)在有機(jī)溶劑里遷移率的不同疏水色譜：根據(jù)蛋白質(zhì)的疏水性質(zhì)第五十五頁(yè)，共六十五頁(yè)，編輯于2023年，星期六凝膠過(guò)濾法：蛋白質(zhì)分子量的對(duì)數(shù)和洗脫體積之間成線性關(guān)系（反比例）SDS-聚丙烯酰胺凝膠電泳法：SDS與蛋白質(zhì)結(jié)合使其帶負(fù)電荷，電泳結(jié)果只與分子量有關(guān)，與所帶電荷無(wú)關(guān)，分子量的對(duì)數(shù)與遷移率之間有線性關(guān)系（反比例）超速離心法：利用蛋白質(zhì)密度不同，經(jīng)超速離心后，分布于不同的液層而分離，蛋白質(zhì)的分子量與其沉降系數(shù)S成正比分子量測(cè)定的方法第五十六頁(yè)，共六十五頁(yè)，編輯于2023年，星期六蛋白的純度和均一性鑒別標(biāo)準(zhǔn)蛋白質(zhì)在SDS呈現(xiàn)一條分離帶等電聚焦電泳呈現(xiàn)一條分離帶在超速離心場(chǎng)中，以單一的沉降速度運(yùn)動(dòng)在色譜分析中呈現(xiàn)一個(gè)洗脫峰經(jīng)純化后的蛋白質(zhì)不失活性第五十七頁(yè)，共六十五頁(yè)，編輯于2023年，星期六紫外吸收法：A280吸收值福林酚法（Folin法）：又稱(chēng)Lowry法，蛋白質(zhì)與福林試