Cell-free synthesis of functional antibody fragments to provide a structural basis for antibody

19 Pages·2017·10.09 MB·English

RESEARCHARTICLE
Cell-free synthesis of functional antibody
fragments to provide a structural basis for
antibody–antigen interaction
TakayoshiMatsuda1☯,TakuhiroIto1☯,ChieTakemoto1,KazushigeKatsura1,2,
MarikoIkeda1,2,MotoakiWakiyama1,2,MutsukoKukimoto-Niino1,2,
ShigeyukiYokoyama2,3,YoshikazuKurosawa4,MikakoShirouzu1,2*
1 DivisionofStructuralandSyntheticBiology,RIKENCenterforLifeScienceTechnologies,1-7-22Suehiro-
cho,Tsurumiku,Yokohama,Japan,2 RIKENSystemsandStructuralBiologyCenter,1-7-22Suehiro-cho,
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Tsurumiku,Yokohama,Japan,3 RIKENStructuralBiologyLaboratory,1-7-22Suehiro-cho,Tsurumiku,
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Yokohama,Japan,4 InnovationCenterforAdvancedMedicine,FujitaHealthUniversitySchoolofMedicine,
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Toyoake,Aichi,Japan
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a1111111111 ☯Theseauthorscontributedequallytothiswork.
*[email protected]
Abstract
OPENACCESS
Growingnumbersoftherapeuticantibodiesofferexcellenttreatmentstrategiesformany
Citation:MatsudaT,ItoT,TakemotoC,KatsuraK,
IkedaM,WakiyamaM,etal.(2018)Cell-free diseases.Elucidationoftheinteractionbetweenapotentialtherapeuticantibodyanditstar-
synthesisoffunctionalantibodyfragmentsto getproteinbystructuralanalysisrevealsthemechanismofactionandoffersusefulinforma-
provideastructuralbasisforantibody–antigen
tionfordevelopingrationalantibodydesignsforimprovedaffinity.Here,wedevelopeda
interaction.PLoSONE13(2):e0193158.https://
rapid,high-yieldcell-freesystemusingdialysismodetosynthesizeantibodyfragmentsfor
doi.org/10.1371/journal.pone.0193158
thestructuralanalysisofantibody–antigencomplexes.Optimalsynthesisconditionsoffrag-
Editor:SalvatoreV.Pizzo,DukeUniversitySchool
ments(FvandFab)oftheanti-EGFRantibody059–152wererapidlydeterminedinadayby
ofMedicine,UNITEDSTATES
usinga30-μl-scaleunit.Theconcentrationofsupplementeddisulfideisomerase,DsbC,
Received:November21,2017
wascriticaltoobtainingsolubleantibodyfragments.Theoptimalconditionsweredirectly
Accepted:February5,2018
applicabletoa9-ml-scalereaction,withlinearscalableyieldsofmorethan1mg/ml.Analy-
Published:February20,2018 sesofpurified059-152-FvandFabshowedthatthecell-freesynthesizedantibodyfrag-
Copyright:©2018Matsudaetal.Thisisanopen mentsweredisulfide-bridged,withantigenbindingactivitycomparabletothatofclinical
accessarticledistributedunderthetermsofthe antibodies.Examinationofthecrystalstructureofcell-freesynthesized059-152-Fvincom-
CreativeCommonsAttributionLicense,which
plexwiththeextracellulardomainofhumanEGFRrevealedthattheepitopeof059-152-Fv
permitsunrestricteduse,distribution,and
broadlycoverstheEGFbindingsurfaceondomainIII,includingresiduesthatformedcritical
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited. hydrogenbondswithEGF(Asp355EGFR,Gln384EGFR,H409EGFR,andLys465EGFR),sothat
theantibodyinhibitedEGFRactivation.Wefurtherdemonstratedtheapplicationofthecell-
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformation freesystemtosite-specificintegrationofnon-naturalaminoacidsforantibodyengineering,
filesexceptapdbfile.Thepdbfileisavailablefrom whichwouldexpandtheavailabilityoftherapeuticantibodiesbasedonstructuralinformation
thePDBdatabase(accessionnumber5XWD).
andrationaldesign.Thiscell-freesystemcouldbeanidealantibody-fragmentproduction
Funding:Theworkwassupportedbythe platformforfunctionalandstructuralanalysisofpotentialtherapeuticantibodiesandfor
following:MinistryofEducation,Culture,Sports,
engineeredantibodydevelopment.
ScienceandTechnology,Japan,http://www.mext.
go.jp/en/,toRIKENCenterforLifeScience
Technologies;MinistryofEducation,Culture,
Sports,ScienceandTechnology,Japan,http://
www.mext.go.jp/en/,TargetedProteinsResearch
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Cell-freesynthesisoffunctionalantibodyfragmentsforstructuralanalysis
ProgramtoShigeyukiYokoyama;Ministryof Introduction
Education,Culture,Sports,Scienceand
Antibodytherapyofferseffectivetreatmentstrategiesformanydiseaseswithahighlyspecific
Technology,Japan,http://www.mext.go.jp/en/,The
PlatformProjectforSupportingDrugDiscovery bindingofamonoclonalantibodytoitstargetantigenprotein,whenitsinactivationleadsto
andLifeScienceResearch(PlatformforDrug thesuppressionofthedisease[1].Elucidationoftheinteractionbetweenapotentialtherapeu-
Discovery,Informatics,andStructuralLife ticantibodyanditstargetproteiniscrucialtounderstandthemechanismsofactionofthese
Science)toShigeyukiYokoyama.Thefundershad
antibodiesandtodeveloprationalantibodydesignforimprovedaffinity[2].Epidermal
noroleinstudydesign,datacollectionand
growthfactorreceptor(EGFR)isoneofthemajortargetsofcancertreatment,because
analysis,decisiontopublish,orpreparationofthe
increasesintheexpressionandactivationlevelsofEGFRarefrequentlyfoundinseveralmeta-
manuscript.
staticcancers[3].EGFRiscomposedofthreesegments:theextracellulardomain(ECD),the
Competinginterests:Theauthorshavedeclared
singlemembrane-spanningregion,andthecytoplasmictyrosinekinasedomain.Epidermal
thatnocompetinginterestsexist.
growthfactor(EGF)bindingtotheECDinducesdimerizationofEGFR,whichleadstoactiva-
tionofthecytoplasmictyrosinekinasedomainandsubsequentstimulationofcellgrowthand
differentiation[4].ManytherapeuticantibodiestargetingEGFRexpressedintumorsarein
clinicaluseorunderdevelopment.Examinationofthecrystalstructuresofanti-EGFRanti-
bodiesincomplexwithEGFRhasrevealedtheirdetailedmechanismsofinhibition.Forexam-
ple,theantigen-bindingfragment(Fab)ofcetuximaborIMC-11F8bindstotheEGF-binding
siteondomainIIIofEGFR,therebypreventingEGF-inducedactivation[5,6].Thematuzu-
mabFabfragmentalsointeractswithdomainIIIofEGFR,whereapartfromtheEGF-binding
site,anditstericallyblocksthedomainrearrangementofEGFRtotheEGF-bindingconforma-
tion[7].059–152isoneofthefullyhumananti-EGFRantibodiesisolatedfromahumansin-
gle-chainvariablefragment(scFv)phagedisplaylibrary[8].The059–152antibodyisa
promisingcandidateforuseinEGFR-targetedantibodytherapy,becauseithaspotentantitu-
moractivityagainstA431epidermoidcarcinomacells.Although059–152IgGinhibitsthe
bindingofEGFtoEGFRaswellasthephosphorylationofEGFR,themolecularmechanismis
notwellunderstood.
Thecell-freeproteinsynthesissystemhasbecomeastandardproteinproductionmethod,
anditprovidesseveraladvantagesoverconventionalcell-basedexpressionmethods[9,10].
Becausethecell-freesystemisanopensystemandfreefromcellviability,thesystemoffers
greatflexibilityfordirectlymanipulatingthereactionenvironment.Forexample,supplement-
ingcell-freereactionswiththeappropriateamountofzincionsignificantlyincreasesthesolu-
bilityandstabilityofzinc-bindingproteins[11].Functionalmembraneproteinscanbe
producedinthepresenceoftheappropriateconcentrationsofspecificdetergentsandlipids
[12].Adialysis-modecell-freesystemcanproducemilligramquantitiesoftheproteinofinter-
est,whichissufficientformanyresearchpurposes,includingfunctionalandstructuralanalysis
[13].Labeledproteiniseasilyproducedbysimplyreplacingnaturalaminoacidswithlabeled
ones,whichisusefulforstructuralanalysisbyX-raycrystallographyandnuclearmagneticres-
onancespectroscopy[14–16].Site-specificlabelingofaproteinwithnon-naturalaminoacids
isaccomplishedbysupplementingwithtemplateDNAcontainingUAGcodonsatthedesired
positions,aminoacyl-tRNAsynthetase,UAG-readingtRNA,andnon-naturalaminoacid[17,
18].
AFabisfrequentlyusedforthestructuralanalysisofantibody–antigencomplexesbyX-ray
crystallography,becausetherigidanduniformstructureoftheFabisbeneficialforcrystal
packing[19].MammaliancellsareusuallyusedtoexpressIgGorFabs,becauseeukaryotic
chaperonesandotherfoldingcatalystsareinvolvedinfacilitatingtheirfolding.AFabispre-
paredbyenzymaticdigestionofIgGbypapain,whichisexpressedfromhybridomacellsor
transientexpressioninmammaliancells.However,theoptimizedparametersforsmall-scale
expressionofIgGorFabsoftendonotscaleuplinearly,andinsuchcasestime-consumingre-
optimizationisrequired[20].Inaddition,inefficientdigestionofsomeIgGsoftenhindersFab
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Cell-freesynthesisoffunctionalantibodyfragmentsforstructuralanalysis
preparation.Bacterial-cell-basedexpressionsystemsareroutinelyusedforscFvandFabprepa-
rationandarestilladvantageousinthattheyareaffordableandenableeasymanipulation,
althoughtheproductionofcorrectlyfoldedeukaryoticproteinsisoftenchallenging,particu-
larlywhentheproteincontainsmultipledisulfidebonds[21,22].Theflexibilityofthecell-free
system,whichallowsdirectmanipulationofthereactionenvironment,makesthesystemsuit-
ableforthesynthesisofdisulfide-containingproteins.Bycontrollingtheoxidizingenviron-
menttopromoteappropriatedisulfidebondformation,thecell-freesystemcanreportedlybe
usedtosynthesizepharmaceuticaldisulfide-containingproteins,includingantibodyfrag-
ments,withouttheneedforalaboriousrefoldingprocess[23–26].Althoughseveralgroups
havereportedcell-freesynthesisofantibodyfragmentsorIgG,applicationofthecell-freesys-
temtostructuralanalysisbyX-raycrystallographyhasnotyetbeendemonstrated[27–29].
Thisremainschallenging,becauseX-raycrystallographyrequiresmilligramquantitiesof
structurallyhomogeneousantibodyfragmentstoobtainwell-diffractedcrystalsthroughcrys-
tallizationscreening.
Here,wedevelopedamethodofpracticalandrapidantibodyfragmentsynthesisforstruc-
turalanalysis.Weshowthatadialysis-modecell-freesystembasedonEscherichiacoli(E.coli)
S30extractisapowerfultoolforproducingantibodyfragmentsforstructuralanalysis,includ-
ingbyX-raycrystallography.Weusedtheanti-EGFRantibody059–152todemonstratethat
milligramquantitiesoffunctionalFvsandFabscanbesynthesized.Furthermore,wesuccess-
fullydeterminedthecrystalstructureofthe059–152FvincomplexwithhumanEGFR-ECD,
whichrevealedanovelmannerofinteractionbetweenanti-EGFRantibodyandEGFRand
providedthestructuralbasisforEGFRinhibitionby059–152antibody.Asfarasweknow,
thisisthefirstevidencethatacell-freesystemcanbeusedtosynthesizefunctional,fully
humanantibodyfragmentsandthatcell-freesynthesizedantibodyfragmentscanbeusedfor
samplepreparationforX-raycrystallography.Moreover,wedemonstratethatthesite-specific
fluorescentlabelingof059–152antibodyfragmentsispossiblewithoutimpairingantigen
bindingactivity.
Materialsandmethods
Plasmidconstruction
ThegenesencodingtheVHCH1andlightchainsof059-152-antibodyweresynthesizedbya
genesynthesisservice(EurofinsOperon,Germany)withcodonusageoptimizationforbacte-
rialexpression.Thenucleotidesequencesencodingthe059-152-VHandthe059–152light
chain(LC)havebeendepositedinGenBank(accessionnumbersLC307162andAB064177.1,
respectively).ThenucleotidesequenceencodingtheCH1domainofhumanIgG1wasusedfor
thatencodingVHCH1,inaccordancewiththeliterature[8].Wesubclonedthegenesencod-
ing059-152-VH,059-152-VL,059-152-VHCH1,andthe059-152-LCintothepCR2.1vector
byusingatwo-stepPCRmethod.TheDNAsequencesencodingtheT7promoter,ribosome-
bindingsite,N11-tag,andtobaccoetchvirus(TEV)proteaserecognitionsitewereattachedat
the50-endofthecodingsequence,whereasthatfortheT7terminatorwasattachedatthe30-
end[30].Notethatsignalpeptidewasexcludedfromthecodingsequence.Theresultingplas-
midswerenamedpCR2.1-059-152-VH,pCR2.1-059-152-VL,pCR2.1-059-152-VHCH1,and
pCR2.1-059-152-LC.
Cell-freesynthesisof059-152-FvandFab
Thedialysismodeofcell-freesynthesiswasusedtosynthesize059-152-FvandFab.Asmall-
scaledialysisunit(30μlinternaland300μlexternalsolution)wasusedtooptimizethereac-
tionconditions[11].Thecompositionoftheinternalsolutionwas60mMHEPES-KOHbuffer
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Cell-freesynthesisoffunctionalantibodyfragmentsforstructuralanalysis
(pH7.5)containing200mMpotassiumL-glutamate,15mMmagnesiumacetate,1.5mMeach
aminoacid,1.3mMATP,0.9mMGTP,0.9mMCTP,0.9mMUTP,81μMfolinicacid,27.6
mMammoniumacetate,80mMcreatinephosphate,1mMreducedglutathione(GSH),4mM
oxidizedglutathione(GSSG),175μg/mltRNAs,0.2to0.8mg/mldisulfideisomerase(DsbC),
100μg/mlcreatinekinase,60μg/mlT7RNApolymerase,30%(v/v)E.coliS30extract,and
2μg/mleachtemplateDNA.ToproducetheFv,pCR2.1-059-152-VHandpCR2.1-059-
152-VLwereaddedtotheinternalsolution.TheplasmidspCR2.1-059-152-VHCH1and
pCR2.1-059-152-LCwereaddedtoproducetheFab.VariableconcentrationsofE.coliDsbC
wereaddedtotheinternalsolutiontodetermineitsoptimalconcentration.Theexternalsolu-
tionwascomposedofthesamecomponentsastheinternalsolutionexceptthatDsbC,creatine
kinase,T7RNApolymerase,andtemplateDNAswereomitted.Inaddition,dithiothreitol-free
S30bufferwasusedinsteadofS30extract[26].Thecell-freereactionwasperformedat25˚C
for7h,unlessotherwisenoted.
Preparationof059-152-FvandFabforX-raycrystallography
Large-scalepreparationof059-152-Fvwasperformedasfollows.059-152-Fvwassynthesized
byusingalarge-scaledialysisunit(9mlinternaland90mlexternalsolution,hereafterreferred
toas9–90mlscale)inthepresenceof1mMGSH,4mMGSSG,and0.2mg/mlDsbC.After
thecell-freesynthesisreaction,theinternalsolutionwasdilutedwithfourtimesthevolumeof
bufferA[20mMTris-HClbuffer(pH7.5)containing500mMNaCland20mMimidazole]
andthencentrifugedat4,500×gfor5min.Afterbeingpassedthrougha0.45-μmfilterunit,
thesupernatantwassubjectedtoaffinitychromatographyusingaHisTrapHPcolumn(GE
Healthcare,USA),andthe059-152-Fvwaselutedwithalineargradientof0%to100%buffer
B[20mMTris-HClbuffer(pH7.5)containing500mMNaCland500mMimidazole].The
elutedfractionswerepooledanddialyzedagainstbufferAbyusingamembranewithamolec-
ularweightcutoff(MWCO)of3.5kDa.TocleavetheN11tag,20μg/mlTEVproteasewas
addedtothesolution,whichwasthenincubatedat30˚Cfor3h.Thecleavedtagwasremoved
bypassingthesolutionthroughaHisTrapHPcolumn.Theflow-throughfraction,containing
059-152-Fv,wascollectedandconcentratedbyusingacentrifugalfilterunitwithaMWCOof
3.5kDa(Millipore,USA).059-152-Fvwasfurtherpurifiedbysizeexclusionchromatography
(SEC)usingaHiload16/60Superdex200columnequilibratedwithbufferC[20mMTris-HCl
buffer(pH7.5)containing150mMNaCl].Theconcentrationofpurified059-152-Fvwas
determinedbyUVabsorbanceat280nm.059-152-Fabwassynthesizedinthepresenceof1
mMGSH,4mMGSSG,and0.8mg/mlDsbC.059-152-Fabwaspurifiedbyusingthesame
procedureasfor059-152-Fv,withminormodifications:adialysismembraneandacentrifugal
filterunitwithaMWCOof10kDawereused.
PreparationofEGFR-ECD
C-terminallyHis-taggedEGFR-ECD(1–643)wastransientlyoverexpressedinHEK293S
GnTI-cells[31].ThesecretedC-terminallyHis-taggedEGFR-ECDwasrecoveredfromthe
mediumbybeingpassedthroughaHisTrapHPcolumn.Afterthecolumnhadbeenwashed
withbufferA,theEGFR-ECDwaselutedwithalineargradientof0%to100%bufferB.The
elutedfractionsweredialyzedagainstbufferA.TheHis-tagwasthencleavedbyTEVprotease
andremovedbyaHisTrapHPcolumn.TheN-linkedoligosaccharidesoftheEGFR-ECD
werecleavedbyendoglycosidaseHtreatment.TheEGFR-ECDwasbuffer-exchangedbydialy-
sisinto20mMTris-HClbuffer(pH8.5)containing10mMNaCl.TheN-linkedoligosaccha-
ridescleavedEGFR-ECDwasseparatedfromtheEGFR-ECDwiththeremainingextra
oligosaccharide(s)onananionexchangecolumn(HiTrapQHP)byusingalineargradientof
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Cell-freesynthesisoffunctionalantibodyfragmentsforstructuralanalysis
0%to25%solutionof20mMTris-HClbuffer(pH8.5)containing500mMNaCl.Themain
peakwasconcentratedbyusingacentrifugalfilterunitandpurifiedbySECusingaHiload16/
60Superdex200columnequilibratedwithbufferC.TheconcentrationofpurifiedEGFR-ECD
wasdeterminedbyUVabsorbanceat280nm.
TopreparecomplexesofEGFR-ECD•059-152-FvandEGFR-ECD•059-152-Fabforcrystal-
lization,theantibodyfragmentsweremixedwithEGFR-ECDatamolarratioof1.2:1.The
complexwasseparatedfromexcessantibodyfragmentsbyusingtheSECcolumnequilibrated
with20mMTris-HClbuffer(pH8.5)containing150mMNaCl.Fractionscontainingthe
complexwereconfirmedbySDS-PAGEandwerethenconcentratedto15mg/mlbyusinga
centrifugalfilterunit.
Crystallization,datacollection,andrefinement
Crystalsofthe059-152-Fv•EGFR-ECDcomplexwereobtainedbyusingthesittingdropvapor
diffusionmethodfromadropcontainingequalvolumesof059-152-Fv•EGFR-ECDsolution
andareservoirsolution[0.1MMESbuffer(pH6.0)containing0.1MZn(OAc) and6%(w/v)
2
PEG8000].Thecrystalswereimmersedstepwiseinreservoirsolutionscontainingincreasing
concentrationsofglycerol[15%,20%,25%,and30%(v/v)].Thecrystalswerethenflashcooled
inliquidnitrogen.X-raydiffractiondatawerecollectedatthePhotonFactorybeamlineAR-
NE3A(Ibaraki,Japan).ThedatasetwasprocessedbyusingHKL-2000software[32].Theini-
tialphasewasdeterminedwithmolecularreplacementmethodusingthesoftwarePhaser[33].
Asasearchmodel,thecrystalstructuresoftheextracellulardomainofhumanEGFR-ECD
[ProteinDataBank(PDB)IDcode:3NJP]andtheFvregionofanti-EGFRFab(PDBIDcode:
3B2U)wereused.ThemodelwasrefinedwiththesoftwareCootandPhenix[34,35].
Kineticanalysis
BindinganalysiswasperformedwithaBIAcore3000(GEHealthcare).TheEGFR-ECDwas
immobilizedonaCM5sensorchipbyusinganaminecouplingkit(GEHealthcare).Kinetic
analyseswereperformedusingsingle-cyclekineticanalysisbymeasuringfivedifferentcon-
centrationseries(50.0,16.6,5.55,1.85,and0.617nM)of059-152-Fvor059-152-Fabineach
cycle.Sensorgramswereobtainedataflowrateof30μl/min,witha60-sassociationphaseand
a120-sdissociationphaseat25˚CinHBS-EP+buffer.Aftereachcycle,thesensorsurfacewas
regeneratedwithaninjectionof10mMglycine-HCl(pH2.4).Kineticparametersweredeter-
minedwithBIAevaluationsoftware(GEHealthcare).Allkineticdataaretheresultsoftripli-
cateexperiments.
Preparationofsite-specificfluorescent-labeledantibodyfragments
WeusedinversePCRtoinserttheDNAsequenceencodingtheGSSGSSlinkerandanamber
codonintotheplasmidspCR2.1-059-152-VHandpCR2.1-059-152-VHCH1justbeforethe
stopcodontoproducepCR2.1-059-152-VH-GSSGSS-amberandpCR2.1-059-152-VHCH1-
GSSGSS-amber.Tosynthesizesite-specificp-azido-L-phenylalanine(AzF)-incorporatedanti-
bodyfragments,9–90mlscalecell-freereactionwasperformedinthepresenceof0.2mg/ml
AzFRS(orthogonalaminoacyl-tRNAsynthetasespecificforAzF),2mMAzF,andS30extract
preparedfromE.colistrainB-95.ΔAharboringtheUAG-readingtRNA-expressingplasmid
pACYC184-piodoTyrRS-MJR1[36].ForthesynthesisofC-terminallyAzF-incorporatedFv,
thetemplateDNAsofpCR2.1-059-152-VH-GSSGSS-amberandpCR2.1-059-152-VLwere
addedtotheinternalsolution,whereaspCR2.1-059-152-VHCH1-GSSGSS-amberand
pCR2.1-059-152-LCwereusedtosynthesizeAzF-incorporatedFab.AzF-incorporatedFvand
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Cell-freesynthesisoffunctionalantibodyfragmentsforstructuralanalysis
Fabwerepurifiedasdescribedabove.S30extractofstrainB-95.ΔAwaspreparedasdescribed
intheliterature[37].
AzF-incorporated059-152-FvandFabweresite-specificallyfluorescentlabeledwithAlexa-
488DIBOalkyne(ThermoFisherScientific,USA)byusingcopper-freeclickchemistry.Using
arotator,20μMantibodyfragmentsweregentlymixedwith200μMAlexa-488DIBOalkyne
for16hatroomtemperatureinPBSbuffer.Alexa-488conjugatedFvandFabwereisolated
fromtheresidualfluorescentdyeinaPD-10columnequilibratedwithPBSbuffer.Labeling
efficiencyandproteinconcentrationwereevaluatedbyusingtheproteinandlabelmethodof
NanoDropspectrophotometers(ThermoFisherScientific).Fluorescenceofthesite-specifi-
callyAlexa-488-conjugated059-152-Fvand059-152-Fabwasconfirmedbyreducingandnon-
reducingSDS-PAGE,asfollows.Homogeneouslypurified0.5μgaliquotsofeachof059-
152-Fv,AzF-incorporated059-152-Fv,andAlexa-488-conjugated059-152-Fv,aswellas059-
152-Fab,AzF-incorporated059-152-Fab,andAlexa-488-conjugated059-152-Fab,was
resolvedon10%to20%gradientgels.Fluorescentgelimageswereacquiredbyusingthefluo-
rescencemethod(excitationwavelength,460nm;emissionfilter,Y515)ofanImageQuant
LAS-4000(GEHealthcare).ThesamegelswerethenstainedwithCoomassieBrilliantBlue
(CBB),andimageswereobtainedbyusingthedigitizingmethodoftheLAS-4000.
Results
Cell-freesynthesisof059-152-Fvand059-152-Fab
AFvcontainstwopairsofdisulfidebonds,andaFabcontainsfivepairs.Inmanyextracellular
proteins,disulfidebondformationandfoldingarecloselycoupledprocesses.Wethereforeco-
translationallyfacilitatedappropriatedisulfidebondformationforthesynthesisoffunctional
antibodyfragments.Cell-freesynthesiswasperformedunderpreciselycontrolledoxidative
conditionsinthepresenceof1mMGSHand4mMGSSGtopromotedisulfidebondforma-
tion.Inaddition,bacterialdisulfideisomerase,DsbCwasintroducedtothecell-freereaction
toreshuffleincorrectlyformeddisulfidebonds.059-152-Fvwassynthesizedinthepresenceof
threedifferentconcentrationsofDsbC(0,0.2,or0.4mg/ml).059-152-VHand059-152-VL
weremostlysolubleunderallthreeconditions.Thesolubilityof059-152-VHwasincreased
slightlybyaddingmorethan0.2mg/mlDsbC(Fig1A).WhensynthesizedwithoutDsbC,par-
tiallypurified059-152-Fvshowedseveralbandsonnon-reducingSDS-PAGE(Fig1B).This
waslikelytohavebeen059-152-Fvthatcontainedincorrectlyformeddisulfidebond(s)and
wasthusinactive.Therefore,toobtaincorrectlydisulfide-bonded059-152-Fv,DsbCneededto
beaddedataconcentrationofatleast0.2mg/ml.Next,059-152-Fabwassynthesizedunder
fourdifferentconcentrationseriesofDsbC(0,0.2,0.4,or0.8mg/ml).Whereasthesolubility
ofthelightchainwasindependentofsupplementationwithDsbC,thesolubilityofthe
VHCH1chainwasstronglydependentontheDsbCconcentration(Fig1C).Themaximum
yieldofsolubleFabwasobtainedwhenDsbCwassupplementedat0.8mg/ml,asjudgedfrom
thebandinnon-reducingSDS-PAGEofpartiallypurifiedFab(Fig1D).Formationofinter-
chaindisulfidebondbetweentheVHCH1chainandthelightchainwasconfirmedbycom-
paringthereducingandnon-reducingSDS-PAGEimages.Therefore,wedeterminedtheideal
synthesisconditionsforFvandFab(DsbCat0.2mg/mlforFvand0.8mg/mlforFab),based
ontheirbandpatternsandintensitiesonthenon-reducingSDSpolyacrylamidegel,aswellas
theyieldsofthesolubleproducts.Underidealconditions,approximately1mgofFvorFab
wassuccessfullysynthesizedfrom1mlofinternalsolution.Interestingly,incontrastwiththe
cell-freesynthesesofVLandlightchainalone,whichweresolubleintheabsenceofDsbC,the
VHandVHCH1chainsaloneformedinsolubleaggregates(Fig1Aand1C).Thisindicated
thatreshufflingofincorrectdisulfidebondsbyDsbCwasanessentialstepforcorrectfolding
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Cell-freesynthesisoffunctionalantibodyfragmentsforstructuralanalysis
Fig1.SDS-PAGEanalysisofcell-freesynthesized059-152-Fvand059-152-Fab.(A)059-152-Fvwassynthesizedunderaseriesofdifferent
concentrations(0,0.2,and0.4mg/ml)ofDsbC,asindicated.Total(T)andsoluble(S)fractionsoftheinternalsolutionwereanalyzedby
reducingSDS-PAGE.(B)Purified059-152-Fvwasanalyzedbyreducingandnon-reducingSDS-PAGE.Theyields(mgper1mlinternal
solution)ofpartiallypurifiedFvareindicatedundereachlaneofthenon-reducingSDSpolyacrylamidegelimage.(C)059-152-Fabwas
synthesizedinthepresenceof0,0.2,0.4,and0.8mg/mlofDsbC,asindicated.(D)Purified059-152-Fabwasanalyzedbyreducingandnon-
reducingSDS-PAGE.Theyields(mgper1mlinternalsolution)ofpartiallypurifiedFabareindicatedundereachlaneofthenon-reducingSDS
polyacrylamidegelimage.BG:cell-freesynthesiswithouttemplateDNA.VH:cell-freesynthesisofVHwithoutDsbC.VL:cell-freesynthesisof
VLwithoutDsbC.VHCH1:cell-freesynthesisofVHCH1withoutDsbC.Lchain:cell-freesynthesisoflightchainwithoutDsbC.Gelswere
stainedwithCBB.
https://doi.org/10.1371/journal.pone.0193158.g001
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Cell-freesynthesisoffunctionalantibodyfragmentsforstructuralanalysis
oftheVHandVHCH1chains.ConsideringourfindingsthatsolelysynthesizedVHCH1still
tendedtoaggregateevenwhensupplementedwithasufficientamountofDsbCandthatco-
expressionofthelightchainconsiderablyimprovedthesolubilityofVHCH1(S1FigandFig
1C),wesurmisedthatthelightchainhadchaperone-likeactivitytofacilitateproductivefold-
ingofVHCH1.
Kineticanalysis
Wepreparedpurified059-152-Fvand059-152-Fabfrom9–90mlscalecell-freesynthesis.The
yieldswere10.8mgand14.5mg,respectively.Kineticanalysiswasperformedbysurfaceplas-
monresonancetoverifythebindingactivityofthecell-freesynthesized059-152-Fvand059-
152-FabtoimmobilizedEGFR-ECD.059-152-Fvand059-152-FabboundtotheEGFR-ECD
withK valuesof5.9nMand7.7nM,respectively(Table1).TheseK valuesarecomparable
D D
tothoseoftheFabsofthepharmaceuticalanti-EGFRantibodiescetuximab(2.3nM)and
IMC-11F8(3.3nM)[5].Thek andk values(Table1),aswellasthesensorgrams(S2Fig),
on off
indicatedthat059-152-Fvand059-152-Fabhadthecharacteristicsoffastbindinganddissoci-
ation.Theseanalysesshowedthatthecell-freesystemwascapableofsynthesizingfunctional
antibodyfragments.
Site-specificfluorescent-labeledantibodyfragmentproduction
Geneticincorporationofnon-naturalaminoacidshasawidevarietyofapplicationsinprotein
engineering.B-95.ΔAisanE.colistraininwhichtheUAGcodonisassignedtoanon-natural
aminoacid[36].Thisstrainisconstructedbyreplacing95of273UAGcodonswithoneofthe
othertwostopcodons(UAAorUGA)intheE.coliBL21(DE3)genome.ThentheprfAgene,
whichencodesreleasefactor1,isdeletedfromthegenometoassigntheUAGcodontoanon-
naturalaminoacid.B-95.ΔAshowsactivegrowthcomparabletothatoftheparentalE.coli
BL21(DE3)andisthussuitableforrecombinantproteinproduction.WepreparedS30extract
fromB-95.ΔA,harboringaUAG-reading-suppressortRNAexpressionplasmid,andusedit
forsite-specificincorporationofAzFinto059-152-Fvand059-152-Fabinthecell-freesystem.
TheC-terminalof059-152-VHand059-152-VHCH1waschosenasasuitableincorporation
site,becausetheC-terminalregionofVHandVHCH1isdistantfromtheantigenbindingsite
andislesslikelytointerferewiththefoldingprocessofVHandVHCH1.TheDNAsequence
encodingtheGSSGSSlinkerwasinsertedbeforetheAzFincorporationsitetokeepAzF
slightlyawayfromtheantibodyfragments,whichwouldenhancethelabelingefficiency.
AzF-incorporated059-152-Fvand059-152-Fabweresuccessfullysynthesizedinthecell-
freesystemsupplementedwith0.2mg/mlAzFRSand2mMAzF.Theyieldsofhomogeneously
Table1. Bindingkineticsof059-152-Fvand059-152-FabtotheEGFR-ECD.
kon(105M-1s-1) koff(10−3s-1) KD(nM)
059-152-Fv 24.9±0.8 14.7±0.5 5.9±0.1
059-152-Fv/AzF 23.6±0.1 13.7±0.4 5.8±0.2
059-152-Fv/Alexa-488 25.2±0.6 15.4±0.2 6.1±0.1
059-152-Fab 19.4±0.5 15.0±0.1 7.7±0.1
059-152-Fab/AzF 17.8±0.4 12.5±0.4 7.0±0.1
059-152-Fab/Alexa-488 16.7±0.5 13.3±0.7 8.1±0.2
Associationrate(kon),dissociationrate(koff),andbindingaffinity(KD)of059-152-Fv,059-152-Fab,AzF-
incorporatedFv,AzF-incorporatedFab,Alexa-488conjugatedFv,andAlexa-488conjugatedFabtoEGFR-ECDare
shown.
https://doi.org/10.1371/journal.pone.0193158.t001
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Cell-freesynthesisoffunctionalantibodyfragmentsforstructuralanalysis
Fig2.SDS-PAGEanalysisofsite-specificfluorescent-labeled059-152-Fvand059-152-Fab.Reducingandnon-
reducingSDS-PAGEanalysisof059-152-Fv(A)and059-152-Fab(B).(lane1)059-152-Fv,(lane2)AzF-incorporated
059-152-Fv,(lane3)Alexa-488conjugated059-152-Fv,(lane4)059-152-Fab,(lane5)AzF-incorporated059-152-Fab,
and(lane6)Alexa-488conjugated059-152-Fab.FluorescentimagesandCBB-stainedimageswereacquiredfromthe
samegels.
https://doi.org/10.1371/journal.pone.0193158.g002
purifiedAzF-containing059-152-Fvand059-152-Fabfromthe9–90ml-scalecell-freereactions
were12.5mgand7.6mg,respectively.Weappliedstrain-promotedazide-alkynecycloaddition,
abio-orthogonalchemicalreaction,tothefluorescentdye–antibodyconjugationreaction.After
removingtheresidualfluorescentdye,wemeasuredthelabelingefficiencyanditwas76%for
059-152-Fvand87.9%for059-152-Fab.Reducedandnon-reducedSDS-PAGEanalysisclearly
showedthefluorescenceoftheAlexa-488-conjugated059-152-Fvand059-152-Fab(Fig2).
Notably,inthereducedSDSpolyacrylamidegelimageof059-152-Fv(Fig2A,lane1),theVH
andVLbandswereclosetogetherandlookedlikeasingleband.Inthenon-reducedSDS-PAGE
imagewefoundafaintbandthatmigratedslightlyslowerthantheFab.Thiswaspresumably
duetopartialreductionoftheFabduringSDS-PAGE.KineticanalysisrevealedthattheAzF-
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Cell-freesynthesisoffunctionalantibodyfragmentsforstructuralanalysis
incorporatedandAlexa488-conjugated059-152-Fvand059-152-Fabretainedtheirnative
bindingaffinitiestotheantigen(Table1).
Determinationofthestructureofthe059-152-Fv•EGFR-ECDcomplex
The059-152-Fv•EGFR-ECDcomplexwaspreparedbymixing059-152-FvandEGFR-ECDat
amolarratioof1.2:1,itwasthenisolatedbysize-exclusionchromatography.High-quality
crystalswereobtainedwithareservoircomposedof0.1MMESbuffer(pH6.0)containing0.1
MZn(OAc) and6%(w/v)PEG8000,anddiffractedupto2.9Å.Theinitialphasewasdeter-
2
minedbymolecularreplacementusingthecoordinatesoftheextracellulardomainofEGFR
(PDBIDcode:3NJP)andtheFvregionofanti-EGFRFabantibody(PDBIDcode:3B2U)as
searchmodels.Theasymmetricunitcontainedone059-152-Fv•EGFR-ECDcomplex,andthe
structurewasrefinedupto2.9Å.Thestatisticsofdatacollectionandrefinementareshownin
Table2.
Overallstructureofthe059-152-Fv•EGFR-ECDcomplex
Examinationofthecrystalstructureofthe059-152-Fv•EGFR-ECDcomplexrevealedthat059-
152-FvbindsexclusivelytodomainIIIoftheEGFRinadifferentmannerfromthoseofthe
previouslydeterminedstructuresofantibody•EGFR-ECDcomplexes(Fig3AandS3Fig).The
bindingsiteof059-152-FvonEGFRdomainIIIlargelyoverlapswiththatofEGF(Fig4),
whichisconsistentwithpreviousinformationthat059–152IgGcompeteswithEGFfor
Table2. Datacollectionandrefinementstatistics.
Datacollectionandrefinement 059–152Fv•EGFR-ECDcomplex
Datacollection
Beamline BL-NE3AinPF
Spacegroup P622
5
Celldimensions
a,b,c(Å) 203.4,203.4,113.8
α,β,γ(˚) 90,90,120
Resolution(Å) 50.00–2.9(3.0–2.9)
R 0.098(2.053)
sym
I/σI 39.8(1.8)
Redundancy 21.7(22.1)
Completeness(%) 99.6(100.0)
Refinement
Resolution(Å) 32.5–2.89
No.ofreflections 30595
Rwork/Rfree 0.2768/0.3309
Numberofatoms 6623
B-factors(Å2) 94.5
R.m.s.deviations
Bondlength(Å) 0.003
Bondangles(˚) 0.560
Ramachandranplot
Favored/allowed(%) 93.8/6.3
PDBIDcode 5XWD
Valuesinparenthesesareforthehighest-resolutionshell.
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