anionicwww.dizhi1.com

www.
genscript.
comIntroduction1860CentennialAve.
,Piscataway,NJ08854,USAToll-Free:1-877-436-7274|Tel:1-732-885-9188|Fax:1-732-210-0262|Email:info@genscript.
comSincethemid-1970s,monoclonalantibodies(mAbs)havepioneereddiscoveryanddevelopmentinthefieldsofbiomedicalscience,toxicology,biopharmaceuticalresearch,andbiologicalsciences.
Monoclonalantibodieshavealsobeenproventobeeffectivetherapeutictreatmentsfordiseasessuchascancer,autoimmunedisorders,andbacterialinfections.
Traditionally,theprocessofcreatingamonoclonalantibodycantakeanywherebetween6to8months.
However,duetotheincreasingdemandofobtainingantibodiesfaster,researchershavedevelopedalternativemethodsofproducingantibodies.
Onesuchmethodisusingcommerciallyavailablesystemsandsyntheticgenestoproduceantibodiesrecombinantly.
Recombinantantibodies(rAbs)aremonoclonalantibodiesthatareconstructedinvitrofromsyntheticgenes.
Tobegintheprocess,theheavyandlightchainsoftheantibodyareisolatedandincorporatedintoanexpressionDNAvector.
Theresultingplasmidsarethentransfectedintoahostexpressionsystemandexpressed.
Theresultingrecombinantantibodiescanbeusedinmostofthesameapplicationsastraditionallyproducedmonoclonalantibodies.
Thistechnologyhascuttheproductiontimeofantibodyproductiontimetoabout1-2months,dependingonthemethod.
Recombinantantibodiescanbeproducedinanumberofprokaryoticandeukaryoticsystems.
Thereareprosandconstoeach,butforthepurposeofthisdocument,mammalianexpressionsystemwillbethefocalpoint.
Mammalianexpressionistheidealsystemforstudyingthefunctionofaparticularproteininaphysiologicallyrelevantenvironment.
Thissystemallowsforthehighestlevelofposttranslationalprocessingandfunctionalactivityoftheprotein.
Mammalianexpressionismostcommonlyusedfortheproductionofnativeantibodies,therapeuticantibodies,andantibodiesusedinfunctionalcell-basedassays.
Sinceantibodiescancomeindifferentsizes,formats,foldingstructure,andmore,theoptimizationstrategiesforexpressionmustbeworkedoutforeachindividualantibody.
Threeaspectsarekeyinrecombinantantibodyexpression:quantity,purity,andfunctionality.
Avarietyoffactorssuchaspoorsequencedesignandchoiceofvector,contamination,suboptimalreagentsandexperimentalconditionscanleadtoproteininsolubility,aggregation,ormisfolding,whichultimatelyaffectrecombinantantibodyexpression.
Inthiswhitepaper,wewillfirstdescribethedifferentformatsofrecombinantantibodies,howtodesignyouexpressionvector,transfectionmethodsyoucoulduse,andusingtransientandstableexpressionmethodologiesandcomparingandcontrastingthetwo.
Thenextsectionswilldiscussseveralpathwaysyoucouldmanipulatetooptimizeexpressionforrecombinantantibodiesandpurificationmethodsthatcanbeusedtoincreasepurityandexpression.
Attheend,thereisasummaryoftroubleshootingtechniquesforvariousstagesofexpressioninachartasareferenceguide(Figure1).
www.
genscript.
com6StepstoOptimizeYourRecombinantAntibodyExpressionWhitePaper2860CentennialAve.
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comwww.
genscript.
comChoosinganexpressionhostsystemChoosingbetweenatransientvsstableexpressionDesigninganexpressionvectorChoosingatransfectionmethodforyourplasmidOptimizingyourcellsystemChoosingyourpurificationmethodFigure1:6Stepstoconsiderwhenplanningatransientrecombinantantibodyexpressionexperiment.
WhitePaperStep1:ChoosingYourExpressionHostCellLineThereareanumberofdifferentexpressionsystemsyoucanuseforrecombinantantibodyexpression,bacterial,yeast,insect,mammalian,plant,andcellfreearethemostcommon.
Thiswhitepaperwillfocusonusingmammalianhostcelllines,sincetheyaremorelikelytoexpress,properlyfoldandyieldnative-likepost-translationalmodificationsofsecretedantibodies1.
Glycosylationprofilesfrommammalian-expressedantibodiesarealsothemostconsistentwiththeantibodiesobservedinvivoandarerelativelyhomogeneousinnature,althoughtheremaybeminordifferencesbetweendifferenthosts2,3.
MammaliancelllinestypicallyusedforproteinandantibodyproductionareChineseHamsterOvary(CHO),NS0,BabyHamsterKidney(BHK),andHumanEmbryonicKidney(HEK293).
ThemostpopularchoicesfortransientandstableexpressionareHEK293andCHO,respectively.
Bothcelllinesarewelldocumentedandhavebeenshowntoworkexcellentlyforrecombinantantibodyexpression.
HEK293cellswereisolatedfromhumanembryonickidney4andhavebeenusedashostforsmall-scalerecombinantproteinproductionforover2decades.
Thesecellscanbeecientlytransfectedinsuspensionatlargescaleusingcost-eectivemethodssuchaspolyethylenimine(PEI)orcalcium-phosphate5.
CHOcells:ThesecellsarederivedfromtheovaryoftheChinesehamster.
Theyarethepreferredmammalianhostforbiologicsproductionworldwide,withover70%approvedmonoclonalantibodiesexpressedinthesecells.
3860CentennialAve.
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comwww.
genscript.
comWhitePaperStep2:DesigningYourExpressionVectorAninitialanalysisoftheprimarysequencewilltelltheresearchersifthetargetwillbeachallengetoproduce.
Thereareseveralbioinformaticstoolsavailablethatcanhelpyouwithyourvectordesign.
Onceyouhavedesignedyourtargetandchosenyourexpressionvector,thenextstepistodesignacloningscheme.
Typically,manyresearchersthesedaystendtoadoptparallelizationstrategiestofacilitatehighthroughputcloningandexpression.
Forexample,strategieslikeLigationIndependentCloning(LIC)orrecombination-basedcloning(In-FusionCloning)willallowthegenerationofmultipleexpressionconstructsatonce,forparallelexpressiontesting.
WhileLICisfriendlier,becauseitaddsminimumvectorresiduesandisperceivedasmorefriendlytocrystallography,recombination-basedcloningisalsoaveryefficientmethod.
LigationIndependentCloning(LIC)approachutilizesthe3'→5'exonucleaseactivityofT4DNAPolymerasetocreateoverhangswithcomplementaritybetweenthevectorandinsert.
LICemploysrelativelylongeroverhangswhichformstablejointsbetweenfragments,allowingfortransformationwithoutligationstep.
Owingtoadualpolymerase/exonucleasefunction,T4DNApolymerasecancreateoverhangsofvaryinglengthsbasedonadefinedsequence.
Theannealedvectorisrepairedduringreplication.
Recombination-basedcloningmethodsuchasIn-Fusioncloning,isdesignedforfast,directionalcloningofoneormorefragmentsofDNAintoanyvector.
ThemethodtypicallyusesPCR-generatedDNAfragmentsandlinearizedvectorsthatcontaina15bpoverlapattheirends.
Thisregionisresponsiblefortherecombination.
Withtheadventandaffordabilityofsyntheticgenes,manyscientistschoosetohavetheirgenesdesigned,synthesizedandsubclonedintoanexpressionvector.
OnethatGenScriptoffersisGenSmartTMSmart.
GenScript-theworld'sleadingproviderofmolecularbiologyservices–hasdevelopedGenSmartDesign:asmartconstructdesigninterfacetomakebuildingconstructseasyforresearchersofallexperiencelevelsacrosstheglobe.
GenSmartDesignisdevelopedbasedonapart-drivendesignphilosophyandbackedupwithourproprietaryalgorithmthatintegratesallyouneedtodesignaDNAconstruct.
GenScriptalsooffersacodonoptimizationtoolnamedOptimumGene.
OptimumGeneGeneDesignsystemisaproprietarygeneoptimizationtechnologythatcanalterbothnaturallyoccurringandrecombinantgenesequencestoachievethehighestpossiblelevelsofproductivityinanygivenexpressionsystem.
TheOptimumGenealgorithmtakesintoconsiderationavarietyofcriticalfactorsinvolvedindifferentstagesofproteinexpression,suchascodonadaptability,mRNAstructure,andvariouscis-elementsintranscriptionandtranslation.
Formoreinformationoneitheroftheseservicespleasevisitwww.
genscript.
com.
Step3:DeliveringYourPlasmidYouwillnextneedtodeterminethedeliverymethodforyourplasmid.
Thereareseveraldifferentmethodsyoucanusetointroduceageneofinterestintomammaliancells.
4860CentennialAve.
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comwww.
genscript.
comWhitePaperAllofthesemethodshavebeenshowntosuccessfullyinducegeneexpressionandhavewellestablishedbenefits,butsometimestroubleshootingandoptimizingthesystemcouldcostaresearchvaluabletime.
Withtheadventandaffordabilityofsyntheticgenes,manyscientistschoosetohavetheirgenesdesigned,synthesizedandsubclonedintoanexpressionvector.
Polyethylenimine(PEI)orCalciumPhosphate:Thismethodisthetriedandtrueversionoftransfection,whichutilizeschemicalssuchaspolyethylenimine(PEI)orcalcium-phosphate4,6toincorporatetheplasmidintothecells.
PEIisastablecationicpolymerthatcondensesDNAintopositivelychargedparticlesthatbindtotheanioniccellsurface,whereitislaterendocytosedintothecell6.
Calcium-phosphateworksinasimilarmethodasPEI,butcanhavevariabilityduetoitssensitivitytoslightchangesinpH,temperature,andbuersaltconcentrations,andcanbecytotoxictomanytypesofcellcultures4.
Electroporation:ElectroporationistheprocessofusinganelectricaleldoncellculturesinordertoincreasethepermeabilityofthecellmembranetoallDNAtobeintroducedintothecell7.
Thisprocessishighlyeectivefortransfectingcellsinsuspensionoradherentcultures,butadownsidetoelectroporationisthatrequiresexpensiveequipmentandalsocausesphysicaldamagetothecells8.
RecombinantViralTransduction:Viraltransductionisusuallythepreferredchoiceforcelltypesthatarediculttotransfectornon-dividingcelltypes.
Retroviral,Lentiviral(amorecomplexretroviral),oradenoviralexpressionsystemsarethemostcommonexpressionsystemsused.
Itallowsforstableintegrationofthetransgene,whichleadstothegeneofinterestbeingcontinuouslyexpressedoverrepeatedcelldivisions.
Onekeyfeatureofretroviralandlentiviraltransductionisthattheyproducereplicationdefectiveparticles,whichallowsforthedeliveryofthedesiredsequence,withoutcontinuedviralreplication.
GenScriptoffersacomprehensivegenesynthesisportfoliothatcanassistyouineverystageofgenesynthesis,plasmidpreparation,molecularcloning,andmutagenesis.
Pleasevisit:https://www.
genscript.
com/molecular-biology-service.
htmlStep4:ChoosingbetweenTransientVsStableExpressionMammalianexpressionexperimentscanbeconductedeithertransientlyorstablyincelllinesinordertoexpressyourproteinorantibodyofinterest.
Boththesetransfectionmethodsinvolvegettingtheforeign(target)geneintothecells.
Intransientlytransfectedcells,theforeignDNAdoesnotintegrateintothehostgenomeandassuchitdoesnotreplicateandiseventuallylostthroughcyclesofcelldivisionoverseveraldays.
Transienttransfectionisgoodifyouhaverelativelysmallyieldrequirementsforrecombinantantibody(rAb)andifyouneeditfast(Table1).
TransientgeneexpressionresultsinshorttermrecombinantAbproduction,typically6-10daysfrom5860CentennialAve.
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comwww.
genscript.
comWhitePaperthepointofDNAtransfection.
HEK293cellsnormallyachievehighertransfectionratesandhigheryieldswithPEI,whichisinexpensiveandresultinginloweroverallproductioncosts.
Transientexpressionisidealforrapidproteinproduction,allowingresearcherstheabilitytouseanymammalianexpressionvector,andtogeneratedataquickly.
Instablytransfectedcells,theforeigngenebecomespartofthehostgenomeandisthereforereplicated.
Stabletransfectionalsobeginstransientlybutthroughaprocessofcarefulselectionandamplification,stableclonesaregenerated.
Descendantsofthesetransfectedcells,alsoexpresstheforeigngene,resultinginastablytransfectedcellline.
Becausethestabletransfectionofcellsisalongerandmorearduousprocess,itispracticalforrAbproductiononlargerscales.
DuringtheinitialselectionprocesswhenresearchquantitiesofrAbsaresufficient(10-1000mg),rapidmethodsofrAbproductionarerequiredandsolargescaletransientexpressionisroutinelyused.
Stablecelllinescanalsobeusedoveragaininmanyexperimentsandisideallysuitedforalongexperimentaltimecourse.
Sincetheexpressionvectorhasaselectionmarker(suchasanantibiotic)thatisaddedtothemediaandcouldcausestressuntothecells,itisrecommendedtoperformadose-responsecurvetodeterminetheoptimalconcentration.
ApproachTransientStablepoolStablecelllineProsQuickesttimelineHighertiterthantransientStableproductionConsModeratetiterPoolwilldecayduringrepeatedproductionLongsetuptimeApplicationReagentandearlydrugdiscoveryUsedtoincreasetransientexpressionyieldasareagent.
LargescaleproductionTable1:Prosandconsbetweenthetypesofexpressionmethodsyoucanuseforrecombinantantibodyproduction.
Figure2:Depictionoftheworkowandtimelinebetweentransientandstableexpression.
Isolation,adaptation,banking&expansionProteinproductionExpressionselection&sub-culturing3+Mouths5-7daysTransfectionHigheciencytransfectionProteinproductionExpressionStableexpressionTransientexpression6860CentennialAve.
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comwww.
genscript.
comWhitePaperStep5:OptimizingYourCellSystemTransienttransfectionprotocolsarefairlysimilartooneanotherandhavethesamebasiccomponents.
Inordertoperformasuccessfulexperiment,itisrecommendedtobeginwithmaintaininghealthycells.
Cellsrequireanumberofcomponents(glucose,nutrients,serum,vitamins,etc.
)togrowhealthyandbesusceptibletotransfection.
Onemajorpathwaywerecommendtinkeringwithistheapoptoticpathway.
Apoptosisistheprocessofprogrammedcelldeath.
Itisahighlyregulatedpathwaythatcanbeinducedbytwoways,1)anintrinsic(internal)pathwaytriggersuchascellularstressor2)anextrinsic(external)pathwaytriggersuchassignalsfromothercells9.
Apoptosisincellcultureisusuallycausedbynutrientdepletionand/ormetaboliteaccumulationandcanbecontrolledusingdifferentmethods.
Onemethodistooptimizeyourmediabycontrollingtheaccumulationofmetabolitesbyusingcomponentstoreduceglutamineorbicarbonate.
Thehelpstomodulatetheamountsofammoniaandcarbondioxide,respectively,inyoursystem10.
Anothermethodfortacklingapoptosisismanipulatingcellsinfavorofcellsurvivaltodevelopresistancetoapoptosis.
SomestudieshaveshownthatcellsdevelopingresistancetoapoptosisbyalteringgeneexpressiontofavorcellsurvivalorbymanipulatingtheDNArepairpathway,canincreasegrowthpotentialofcells11.
Manipulationoftheapoptosispathwayisaverypotentwaytohelpoptimizeyourcellsystemforantibodyexpression.
Inordertooptimizetransientantibodyexpressionincelllines,youcanusedifferentcomponentsormethodstoaltertheproliferationorcellgrowthpathway.
Mostcelllinesrequirethecompletegrowthmediatogrow.
However,therearesomeadditionalmediacomponentsthatyoucanuse,suchashormones,growthfactors,andsignalingsubstances,thathavebeenshowntohelpoptimizeyourantibodyexpression.
Oneexampleofagrowthfactor,LONGR3IGF-I,isaengineeredpeptidethathasbeenshowntoenhancecellviabilityovera12dayexperimentinsomecelllinessuchasCHOandHEK29312,13.
Inordertotestwhichtypeofcomponentwouldbegoodforyourcellsystem,youwillneedtodoamediaanalysistest.
Thiswillhelpyoudeterminewhichcombinationofadditiveshelptoincreaseyourantibodyexpression,withminimalchangestoyourcellviability.
Youcanalsoaddadditivestoyourcellculturetohelpoptimizeexpression.
Anexampleofanadditiveishistonedeactylaseinhibitorsthatde-condenseyourchromatinandincreaseyourtranscriptionalactivity.
ProprietaryfeedsolutionssuchasHyCloneCellBoost,canalsoincreaseyourantibodyexpressionbysupplementingessentialcomponentsinyourmediathatnaturallybecomedepletedduringantibodyproduction12.
Overexpressionofdifferentproteins(Aven,Bcl-2,Htert,areafew),canactuallyincreasetheproliferationandsurvivalofsomecelllines14,15.
GenScripthasuseddifferentcomponentsthatregulatecellgrowthforourHighDensity(HD)TransientExpressionHEK293celllinesandhasshownthatusingthecomponentstogetheratthesamepercentage,increasedcellgrowthascomparedtousingthemindividually(Figure3).
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comwww.
genscript.
comWhitePaperFigure3:OptimizationofrecombinantantibodyexpressionusingdierentcellgrowthregulatorsinspecializedGenScriptownedHighDensity(HD)293cells.
ThefollowingresultsshowthatComponent1doesnotworkaloneandComponent2canimproveantibodyexpressionby85%whenaddedtocells.
Control1+25%comp225%comp125%comp21+25%comp21+10%comp20255075HumanIgGtiteratDay5(mg/L)CellRegulatorComponentCombinationsAC-HPLC(%)Anothermajorpathwaythatcanbeadjustedinordertoincreaseproteinexpressionisthecellmetabolismpathways.
Cellularmetabolismpathwaysareinterrelatednetworksandthecharacteristicsofthesepathwayscanhelpwithmediumoptimization.
AnexampleistheglycolysispathwayandtheTCAcycle.
Ifyouhaveacelllinethattendstoproducelactate,itwouldbebeneficialtousecomponentsorchemicalsthatwillleadtoametabolicfluxtowardstheTCAcycle.
Anotherexampleisthatmetabolismcomponentscanalsoimpactantibodyproductionandbiomassformation.
Ifacelllinehasahighgrowthratebutlowproductivity,limitingthemetabolismratescouldforcethecellstoworkonproteinproductioninsteadofbiomass16.
GenScripthastriedseveralmetabolicboosterssuchasenergyboosters,stressreleasers,andproteinfoldingenhancerstooptimizeourtransientexpressionproduction(Figure4).
Thesedifferentboostersneedtobetestedindividually,incombinationwithoneanother,andatdifferentamountswithinyoursystem.
Eachofthemcanactdifferentlyuponthecells,soitisgoodtotesttheminitiallyinsmallamountsandindifferentcombinations.
8860CentennialAve.
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comwww.
genscript.
comWhitePaperControl12345678910111213141516171819202122232425262728020406080IgGTiter(mg/L)ComponentCombinationsDoubledHD293AC-HPLCComponentCocktailofSample18FunctionRatio(%)Component210Component410Component510Component610CellcycleregulatorProteinfoldingenhancerGrowthstressreleaserEnergyboosterFigure4:Optimizationoftransientexpressionusingametabolic,cellcycleandproteinfoldingpathwaymodicationsinspecializedHD-293cells.
Results:Using28dierentpathwaymodicationcombinations,Sample18provedtobethemosteectivechemicalcocktailatincreasingproteinexpression.
Thesampleswerecollectonday5aftertransfection.
Somestudieshaveshownthatinsomesystems,introducingagenethathelpstopromotecellsurvivalbycircumventingthestressresponsepathway17.
Thecellularstresspathwayisacell'sresponsetostressors,suchastemperaturechanges,toxins,environmentstressors,damage,etc.
Inonepaper,scientistsshowedthatbyinducingasmallamountofanexternalstressoruntocellstoinduceastressresponsecanactuallyleadtoaseveralfoldincreaseindifficulttoexpressproteins18.
Anothersystemthatcanbemanipulatedtohelpincreaseproteinexpressionisproteinfolding.
Proteinfoldingisthephysicalprocessbywhichaproteinacquiresitsnativeconformationthatusuallyhelpswithitsbiologicalfunction.
Proteinsrequireproperfoldingtodotheirbiologicalfunction,especiallyproteintherapeuticsthatrequireproperfoldingtoachievethethree-dimensionalconformationinordertobefunctional.
Also,thereareseveraldiseasesthatarecausedbyproteinmisfolding,reiteratingitsimportancenotonlyinresearchbutinbiology.
Studieshaveshownthatfacilitatingtheproperproteinfoldingcanhelpincreaseproteinproduction19.
9860CentennialAve.
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comwww.
genscript.
comWhitePaperFigure5:Genscript'sone-stop-shopplatformfromgenesynthesistopurifiedrecombinantantibody.
TCGCGCGTTTCGGTGATACGGTGAAAACCTCTGAACATCACAGCTTGTCTGTAGCGGATGCCGGGAGCAACAAGCCCGTCAGGGCGCustomerprovidessequenceGenScriptstartswithcodonoptimization,genesynthesisandconstructgenerationExpressionConsrtuctsGeneratedCellCultureandTransfectionAntibodyPuricationandAnalysisFastDeliveryDeliveryofyourrecombinantantibodiesStep6:PurificationTechniquesAnotherstrategytoimproverecombinantantibodyproductionistoimproveyourpurificationmethod.
Recombinantantibodiescanbepurifiedmanuallyorbyusingachromatographysystem.
Dependinguponapplicationandpurityrequirements,thepurificationstepscanbeplannedout.
Iftheantibodyexpressionisrobust,a1-stepaffinitypurificationstepwillyieldsufficientantibodyandadequatepurityforavarietyofapplications.
Iffurtherpurificationisrequired,itcanbetypicallyfollowedwithSizeExclusionChromatography(SEC).
IonExchange(IEX)ChromatographyandHydrophobicInteractionChromatography(HIC)canbeusediffurtherpolishingisrequired.
GenScripthasacomprehensiveproteinexpressionplatform.
Allservicescontainasimilarworkflowbutareadjustedasnecessaryforeachservice.
Thegeneralworkflowisasfollowed:1.
Eachservicebeginswithacustomersuppliedproteinsequenceorcustomer-suppliedDNAvector.
2.
GenScriptthenperformscodonoptimizationusingOptimumGeneTM,GenScript'sPSO-basedproprietarygeneoptimizationtechnology.
Followedbygenesynthesis,plasmidpreparation,andconstructgeneration.
3.
ThegeneofinterestisthensubclonedintoGenScriptproprietaryexpressionplasmid.
4.
Theexpressionplasmidisthentransfectedintotheexpressionsystemofchoice.
Thecellsarethencultured,harvested,andtheproteiniscollected.
5.
Thenthereisone-stepofpurification.
Thepurifiedproteinthenundergoesourstrictqualitycontrolprocess.
6.
Lastly,thepurifiedprotein,DNAconstruct(uponrequest),andQCresultsaredeliveredtothecustomer.
Anitychromatography,separatesproteinsonthebasisofareversibleinteractionbetweenproteinandaspecicligandthatiscoupledtoachromatographymatrix.
Thetechniqueoershighselectivity,highresolution,andhighcapacityfortheproteinofinterest.
Recoveryofpuriedmaterialisgenerallyveryhigh.
10860CentennialAve.
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comwww.
genscript.
comWhitePaperSizeExclusionChromatography(SEC),alsocalledGelFiltration(GF)chromatography.
SECseparatesmoleculesaccordingtodierencesinsizeastheypassthroughtheresin.
Thegoalcanbetoisolateoneortoanalyzethemolecular-weightdistributioninthesample.
SECallowstheusertoincreasepurityaswellashomogeneityoftheantibodysample.
UnlikeinIonExchangeorAnityChromatography,moleculesdonotbindtothechromatographymedium.
IonExchange(IEX)Chromatography,separatesantibodiesonthebasisofdierencesintheirnetsurfacecharge.
IttakesadvantageofthefactthattherelationshipbetweennetsurfacechargeandpHisuniqueforeveryantibody.
Dependingupontheirchargeproperties,antibodiesexhibitdierentdegreesofinteractionwithchargedchromatographymedia.
Dependingontheantibodiesisoelectricpoint(pI),eitheranion-exchangeorcation-exchangematricesareusedinlowsaltconcentrationstobindthetargetproteinswhilecontaminatingproteinsbindrelativelyweakly.
Asthesaltconcentrationisincreased,theinteractionsofchargedgroupsonproteinsurfacebecomesweakerandeventuallyallproteinseluteatacharacteristicsaltconcentrationrangeandcanbeseparated.
HydrophobicInteractionChromatography(HIC):HICseparatesantibodiesaccordingtodierencesintheirsurfacehydrophobicitybyutilizingareversibleinteractionbetweentheseproteinsandthehydrophobicsurfaceofamedium.
Aninversegradientfromhighsalttolowsaltconcentrationisappliedtothecolumn.
Theelevatedsaltconcentrationenhancesinteractionbetweenthehydrophobiccomponentsofthesampleandthechromatographymedium.
Duringseparation,samplesarepuriedandelutedinsmallervolumes,therebyconcentratingthesamplesothatitcangodirectlytogelltrationortoanionexchangeseparation(afterabuerexchange).
HICcanbeusedforcapture,intermediatepuricationorpolishingstepsinapuricationprotocol.
Figure6:SDS-PAGEanalysisofHis-taggedproteinspuriedafterone-steppuricationusingdierentcolumns.
ProteinXisindicatedwithredarrows.
Thecorrespondingexpressionlevelsarelistedinthefollowingtable.
(R)Reducingcondition(N)Non-reducingcondition2001169766442961420kDa25015010075503725201015kDaBeforeOptimizationAfterOptimizationRNRN11860CentennialAve.
,Piscataway,NJ08854,USAToll-Free:1-877-436-7274|Tel:1-732-885-9188|Fax:1-732-210-0262|Email:info@genscript.
comwww.
genscript.
comWhitePaperTroubleshooting:StageExpressionPurificationNoExpressionLowExpressionTruncatedExpressionWrongprotein/antibodypurifiedProtein/AntibodyeluteswithcontaminantsAdditionalPurityNeededProtein/AntibodyprecipitatesProtein/AntibodyelutesduringwashstepsProtein/AntibodydoesnoteluteCommentsProblemChecktomakesureDNAsequenceisOKCodonoptimizationtoenhanceexpressionOptimizeexpressionconditions(media,lowerinductiontemperature,etc.
)EnsureDNAsequenceisOKandthattherearenotprematurestopcodonsTryinductionatlowertemperatureCheckDNAtoensureconstructintegrityBindingandwashconditionsneedtobemorestringent–includeupto20mMimidazoleinbindingandwashbuffersColumntoolarge–ReduceresinamountContaminantsassociatedwithtaggedprotein–addreducingagentsuchasβ-MEtoreducedisulfidebondformationPerformadditionalpurificationstepssuchasIon-ExchangeChromatographyMaintainlowconcentrations5mMDTTtopreventoxidationMaintainhighsaltconcentration500mMandaddglycerol>10%Addarginineintherangeof50mM-500mMAddmildnon-denaturingdetergentsuchas0.
1%β-octylglucosidePerformpurificationatroomtemperatureLowerimidazoleconcentrationinwashbufferReducestringencyofwashbufferElutionconditionsmaybetoomild–elutewithpHorelutionbufferwithstep-gradienttodetermineoptimalelutionconditionsProteinmaybeinaggregate(oligomer/multimer)form–EluteunderdenaturingconditionsProteinhasprecipitatedincolumn–performbindingandelutioninbatchformattoavoidhighproteinconcentrationlocallyTable2:Troubleshootingtabletoassistatdierentstagesoftransientexpressionexperiments.
Summary:Wehavesummarizedvariousstrategiesthatcanimproveyourproteinexpression.
Thesestrategies,whilenotcomprehensive,areofconsiderableimportanceforachievingreliableandhigh-levelproteinexpressionforyourresearch.
PleaseseethefollowinginformationtolearnhowGenScript'servicecanhelpincreaseyourrecombinantantibodyyields.
12860CentennialAve.
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comwww.
genscript.
comWhitePaperHighDensityTransientExpressionSystem:Keepingcustomerneedsinmind,GenScripthaslaunchedanew,HighDensity(HD)TransientExpressionserviceforthehigh-titerproductionofyourrecombinantantibodiesandproteinsineitherCHOorHEK293cells.
UsingHDmammaliancellculture,thisHDTransientExpressionservicecandeliverunprecedentedimprovementsinfunctionalprotein/rAbyieldscomparedtoregularTransientExpressionservices.
Usingthisnewservice,GenScriptscientistscandelivergramquantitiesofintracellularandsecretedproteins(includingantibodies)foryourfunctional,structuralandtherapeuticstudies.
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Dalton,A.
C.
&Barton,W.
A.
Over-expressionofsecretedproteinsfrommammaliancelllines.
ProteinSci.
23,517–25(2014).
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Jenkins,N.
,Meleady,P.
,Tyther,R.
&Murphy,L.
Strategiesforanalysingandimprovingtheexpressionandqualityofrecombinantproteinsmadeinmammaliancells.
Biotechnol.
Appl.
Biochem.
53,73–83(2009).
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Chusainow,J.
etal.
Astudyofmonoclonalantibody-producingCHOcelllines:whatmakesastablehighproducerBiotechnol.
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102,1182–96(20094.
Graham,F.
L.
&vanderEb,A.
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Anewtechniquefortheassayofinfectivityofhumanadenovirus5DNA.
Virology52,456–67(1973).
5.
Durocher,Y.
,Perret,S.
&Kamen,A.
High-levelandhigh-throughputrecombinantproteinproductionbytransienttransfectionofsuspension-growinghuman293-EBNA1cells.
NucleicAcidsRes.
30,E9(2002).
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Longo,P.
A.
,Kavran,J.
M.
,Kim,M.
-S.
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KeyfeaturesofHDTransientExpressionServiceHD-HEKandHD-CHOforchoiceofHEK293orCHOexpressionExpertvectordesignatthemolecularleveltooptimizeantibodyexpressionExtensiveexperiencewithdicultantibodyformats.
Yieldimprovementsrangingupto100foldShortenedturnaroundtime(sequencetoprotein/antibodyinaslittleas10weeks)Upto3g/LrAbtiterpossibleLearnmoreat:https://www.
genscript.
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html13860CentennialAve.
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