TitleThe intervening removable affinity tag (iRAT) productionsystem facilitates Fv antibody fragment-mediatedcrystallography

Author(s) Nomura, Yayoi; Sato, Yumi; Suno, Ryoji; Horita, Shoichiro;Iwata, So; Nomura, Norimichi

Citation Protein Science (2016), 25(12): 2268-2276

Issue Date 2016-12

URL http://hdl.handle.net/2433/216542

Right

This is the accepted version of the following article: [Nomura,Y., Sato, Y., Suno, R., Horita, S., Iwata, S. and Nomura, N.(2016), The intervening removable affinity tag (iRAT)production system facilitates Fv antibody fragment-mediatedcrystallography. Protein Science, 25: 2268‒2276.], which hasbeen published in final form athttp://dx.doi.org/10.1002/pro.3035. This article may be usedfor non-commercial purposes in accordance with Wiley Termsand Conditions for Self-Archiving.; The full-text file will bemade open to the public on 22 November 2017 in accordancewith publisher's 'Terms and Conditions for Self-Archiving'.; この論文は出版社版でありません。引用の際には出版社版をご確認ご利用ください。This is not the published version.Please cite only the published version.

Type Journal Article

Textversion author

Kyoto University

1

Theinterveningremovableaffinitytag(iRAT)

productionsystemfacilitatesFvantibodyfragment-

mediatedcrystallography

YayoiNomura1,2,YumiSato1,2,RyojiSuno1,ShoichiroHorita1,SoIwata1,2,3*,and

NorimichiNomura1,2*

1Department of Cell Biology, Graduate School of Medicine, Kyoto University,

Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan. 2Japan Science and

Technology Agency, Research Acceleration Program, Membrane Protein

Crystallography Project, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.3RIKENSPring-8Center,Kouto,Sayo-cho,Sayo-gun,Hyogo679-5148,Japan

CorrespondingAuthors:

SoIwata

DepartmentofCellBiology,GraduateSchoolofMedicine,KyotoUniversity,

Yoshida-Konoe-cho,Sakyo-ku,Kyoto606-8501,Japan

Phone:81-75-753-4372;Fax:81-75-753-4660

E-mail:s.iwata@mfour.med.kyoto-u.ac.jp

NorimichiNomura

DepartmentofCellBiology,GraduateSchoolofMedicine,KyotoUniversity,

Yoshida-Konoe-cho,Sakyo-ku,Kyoto606-8501,Japan

Phone:81-75-753-4389;Fax:81-75-753-4660

E-mail:nnomura@mfour.med.kyoto-u.ac.jp

2

RunningTitle:

FvantibodyproductionviatheiRATsystem

Manuscript:

-Main-text:27pages

-Supplementarymaterials:1page

SupplementaryFigure1(Filename:iRAT_FigS1.tif)

-Threefigures

-Onetable

3

Abstract:

Fv antibody fragments have been used as co-crystallization partners in

structural biology, particularly in membrane protein crystallography.

However, there are inherent technical issues associated with the large-

scaleproductionofsoluble,functionalFvfragmentsthroughconventional

methodsinvariousexpressionsystems.Tocircumventtheseproblems,we

developedanewmethod,inwhichasinglesyntheticpolyproteinconsisting

of a variable light (VL) domain, an intervening removable affinity tag

(iRAT),andavariableheavy(VH)domainisexpressedbyaGram-positive

bacterialsecretionsystem.Thismethodensuresstoichiometricexpression

ofVLandVHfromthemonocistronicconstruct followedbyproperfolding

and assembly of the two variable domains. The iRAT segment can be

removedbyasite-specificproteaseduringthepurificationprocesstoyield

tag-free Fv fragments suitable for crystallization trials. In vitro refolding

stepisnotrequiredtoobtaincorrectly foldedFvfragments.Asaproofof

concept, we tested the iRAT-based production of multiple Fv fragments,

includingacrystallizationchaperoneforamammalianmembraneprotein

as well as FDA-approved therapeutic antibodies. The resulting Fv

fragments were functionally active and crystallized in complex with the

targetproteins.TheiRATsystemisareliable,rapidandbroadlyapplicable

meansofproducingmilligramquantitiesofFvfragmentsforstructuraland

biochemicalstudies.

Keywords:antibody,Fvfragment,crystallizationchaperone,membraneprotein,

therapeuticantibody,polyprotein,secretoryexpression,Gram-positivebacteria

4

A 50-75-word statement, written for a broader audience, outlining the

importanceand/orimpactoftheworkpresentedinthemanuscript:

Fv antibody fragments are versatile co-crystallization partners to aid the

structural determination ofotherwise'uncrystallizable' proteins. In this study,

we developed a fast and reliable method for production of Fv fragments in

milligramquantities.TheiRATsystemisanaccessiblemethodforuseinabroad

rangeofantibody-mediatedcrystallographyandstructure-basedresearchonthe

binding modes of therapeutic antibodies. Moreover, it may generally be

applicabletothelarge-scaleproductionofantibodyreagentsandtherapeutics.

5

Introduction

Structuralstudiesofkeyproteinsthatareinvolvedinbiologicallyandmedically

relevantmolecularprocessesarefrequentlyabandonedbecauseofdifficultiesin

preparing diffraction-quality crystals. Inmost cases, theirmolecular flexibility,

conformational heterogeneity or polydispersal characteristics in solution

hindersproteincrystallization.Chaperone-assistedcrystallographyisapowerful

strategy to address this problem and can be applied to a broad range of

membrane proteins and otherwise ‘uncrystallizable’ soluble proteins, such as

heavilyglycosylatedandmulti-domainproteins1,2.Thechaperonemoleculesaid

in crystallization by increasing the hydrophilic surface area available for rigid

crystal lattice formation. The bound chaperonemolecules reduce the inherent

protein flexibility and conformational heterogeneity, thereby increasing the

chanceofsuccessfulcrystallizationofdifficulttargetproteins.

Differentproteinscaffoldshavebeenusedascrystallizationchaperones,

rangingfromanimal-derivedantibodyfragments3,4tosyntheticbinderssuchas

DARPins5andMonobodies6.DNAandRNAaptamershavealsobeenappliedas

co-crystallization partners7,8. Among the reported chaperones, Fv antibody

fragmentisapromisingscaffoldthathasbeenusedtocrystallizeofavarietyof

membrane proteins including bacterial cytochrome c oxidase9,10, yeast

cytochrome bc1 complex11, archaeal KvAP voltage-gated potassium channel12,

human adiponectin receptors13 and the mammalian fructose transporter

GLUT514. In addition, the compactness of Fv fragments affords additional

advantagesinstructuralstudiesexaminingtheantigen-antibodyinterface,such

asthecharacterizationoftherapeuticantibodyblockingmechanisms15,16.

6

PreparationofmilligramquantitiesofpurifiedFv fragments is themost

criticalprerequisitewhenFv fragmentsareusedasco-crystallizationpartners.

Several studies evaluating the quantity and quality of Fv fragments produced

from different expression systems have been performed. Bicistronic VL-VH

overexpression in theEscherichiacoliperiplasmresults in relatively lowyields

(0.5-1mgperliterofculture)17.RecoveryoffunctionalFvfragmentsfromE.coli

cytoplasmicinclusionbodiesrequiresalaboriousandtime-consumingrefolding

process during purification18. In contrast, Gram-positive bacteria lack an outer

membraneandhaveapowerfulsecretionapparatus thatallowsdisulfidebond

formation and the proper folding of antibody fragments in an oxidizing

environment. Bicistronic VL-VH secretory expression has been tested in

Streptomyces lividans to purify a certain Fv fragment19, but it is not known

whether this method can be practically applied to a wide range of antibody

clones.ProductionofFvfragmentsbytwo-memberedcultureoftheseparateVL-

and VH-expression strains in Brevibacillus chosinensis secretory system14 is

problematicwheneitherstrainproliferatestoorapidly.

Tosolvetheseproblems,wehavedevelopedanewexpressionstrategyin

which thestoichiometric secretionandassemblyof theVLandVHdomainsare

achieved by monocistronic expression in the Gram-positive bacterium B.

choshinensis. A polyhistidine-maltose binding protein (His6-MBP) cassette,

embeddedwithintheinterveningregionofthetwovariabledomains,servesas

an affinity purification tag that can be removed by tobacco etch virus (TEV)

proteasecleavage.Wechosethename“iRAT(interveningremovableaffinitytag)

system” to describe this production strategy. Here, we present a reliable and

7

broadly applicablemethod for preparingmilligramquantities of Fv fragments.

This method was validated through the production of multiple Fv fragments

derived from murine and humanized monoclonal antibodies including

therapeutic antibodies approved by the U.S. Food and Drug Administration

(FDA). The suitability of the resulting Fv fragments for structural studieswas

alsodemonstratedthroughco-crystallizationandX-raydiffractionanalyses.

Results

ConceptanddevelopmentoftheiRATsystem

We developed the iRAT system to address the need for a reliable method to

controlthe1:1stoichiometricexpressionandtheproperfoldingoftheVLandVH

domains, each ofwhich contains an intrachain disulfide bond. To increase the

throughputofcrystallizationtrials,itisidealtocollectsolubleandfunctionalFv

fragmentswith high yields by using simple purification procedureswithout in

vitro refolding, a time and labor intensive process. Considering these

prerequisites,wegeneratedasyntheticpolyproteinencodingboththeVLandVH

domains by using a monocistronic expression strategy in a Gram-positive

bacterial secretion system. Specifically, the new expression vector pBIM2

containedtheVL-andVH-codingsequencesclonedinasinglereadingframe(Fig.

1A). The two variable domains were tethered together by the iRAT segment,

whichcontainedthefirstTEVcleavagesite,theHis6tag,a linker,MBP,another

linkerandthesecondTEVcleavagesite inthatorder.The iRATportioncanbe

removed by cleavage with His6-tagged TEV protease (TEV-His6) followed by

8

reverse immobilized metal-affinity chromatography (IMAC) (Fig. 1B and

Methods). MBP was included to enhance the secretory expression of the

syntheticpolyprotein.ThepurificationyieldsforiRAT-basedFvexpressionwere

1.5 to 1.7-fold higher than those of the non-MBP fusion. Another reason for

includingMBPisthatthedistancebetweentheN-andC-termini(43Å)issimilar

to the distance between the C-terminus of VL and the N-terminus of VH

(approximately 35-40 Å) for most Fv fragments20. The construct was cloned

downstreamoftheconstitutivelyactivepromoterP5andtheSecsignalpeptide

forpolyproteinsecretionintotheculturesupernatant.

iRAT-based production of a crystallization chaperone for the rat fructose

transporterGLUT5

Inapreviousstudy,anFvfragment(clonenumber:4D111)wasusedtoimprove

thecrystallizationoftheratGLUT5fructosetransporter14.Wetestedthelarge-

scale expression and purification of the Fv 4D111 by using the iRAT system.

BrevibacilluscellsharboringthepBIM2-basedconstructwerecultivatedfor48h.

Afterward, Fv 4D111 was purified from the culture supernatant by using

ammoniumsulfateprecipitationfollowedbyIMAC,TEVcleavage,reverseIMAC

and size exclusion chromatography (SEC) (Methods). The yield of purified Fv

4D111was 4.1mg per liter of culture, and no apparent aggregation occurred

whenthepurifiedproteinwasconcentratedto~10mg/mL.AtypicalSECelution

profileforFv4D111isshowninFigure2A.Theelutionpeakwasmonodisperse,

andoligomerformationwasnotobserved.Calibrationwithmolecularstandards

revealedthattheFv4D111existedasamonomerinsolutionwithanapparent

9

molecularweight of ~26 kDa. SDS-PAGE under reducing conditions showed a

doublebandwiththeexpectedmobilityoftheVLandVHdomains(Fig.2B).

To testwhether thepurifiedFv fragmentwas functional, SECof the rat

GLUT5transporterincomplexwithFv4D111wasperformed.Amolarexcessof

Fv 4D111 was mixed with GLUT5 and loaded onto an SEC column. Complex

formation was indicated by an elution peak shift toward higher molecular

weights,ascomparedwithuncomplexedGLUT5(Fig.2C).SDS-PAGEanalysisof

thehighmolecularweightpeakclearlyshowedthepresenceofbothGLUT5and

Fv4D111(Fig.2D).Surfaceplasmonresonance(SPR)experimentsindicatedthat

iRAT-producedFv4D111boundGLUT5withaKDvalueof2.4nM(Fig.2E).The

thermalstabilityofGLUT5inthepresenceorabsenceofFv4D111indicatedthat

this antibody fragment increased theTmby 12.5°C after GLUT5 binding, thus

suggesting that Fv 4D111-bound GLUT5 was significantly stabilized (Fig. 2F).

Furthermore,theGLUT5-Fv4D111complexwasabletobecrystallized(Fig.2G,

inset).Theco-crystalsdiffractedupto3.2Åandgeneratedcompletediffraction

sets, whichwere able to be processed to solve the structure of the GLUT5-Fv

complex (Fig. 2G).These results suggest that the iRAT system isuseful for the

production of Fv scaffold-based crystallization chaperones in milligram

quantities.

iRAT-based production of Fv fragments of therapeutic antibodies for co-

crystallizationwiththeirtargets

A structure-based understanding of the interactions between therapeutic

10

antibodies and target proteins is critical to the rational design of improved

biotherapeutics.However, a relatively small number of crystal structures have

been determined for therapeutic antibody-target protein complexes, which

probably reflects the lack of simple methods facilitating production of

recombinant antibody fragments in milligram quantities, resulting in limited

success in co-crystallization trials. Typical examples include certolizumab and

denosumab.Certolizumabisahumanizedmonoclonalantibodythattargetsthe

tumor necrosis factorα (TNFα) and is used to treat rheumatoid arthritis and

Crohn’sdisease21.Denosumabisahumanmonoclonalantibodythattargetsthe

receptor activator of nuclear factor κB ligand (RANKL) and is used to treat

osteoporosis, treatment-induced bone loss, metastases to bone, and giant cell

tumorsofthebone22.AlthoughtheU.S.FDAhasapprovedthesetwoantibodies,

theinteractionsbetweentheantibodiesandtheirtargetmoleculeshavenotbeen

accurately mapped at atomic-level resolution by crystallographic analyses. To

demonstrate theutilityof the iRATsystem,weproducedFv fragmentsderived

fromthesetwotherapeuticantibodies.

Amino acid sequence data for the antibodies were retrieved from

DrugBank(http://www.drugbank.ca/).Codon-optimizedcDNAsencodingtheVL

andVHdomainswereartificiallysynthesizedandclonedintothepBIM2vector.

ThecertolizumabFvandthedenosumabFvwerereadilyexpressedandpurified

to greater than95%puritywith yields of 15.4 and1.0mgper liter of culture,

respectively. Excess amounts of each of the purified Fv fragmentsweremixed

withthecorrespondingtargetprotein.Theresultingcomplexesappearedtobe

monodisperse, as indicated by the SEC analysis (Figs. 3A and 3D). SDS-PAGE

11

analysis confirmed that the higher-molecular-weight peak fractions contained

theFvfragmentanditstargetcytokine,thusindicatingtightcomplexformation

betweenthem(Figs.3Band3E).Purified,concentratedcomplexeswereusedto

setupbroadcrystallizationscreens,andthecrystallizationconditionswerethen

optimized by varying the precipitant concentration. Promising crystals whose

complete diffraction data setswere collected are shown in Figures 3C and 3F.

Thedatacollectionstatisticsareshown inTable1.Molecularreplacementwas

performedusingtheFv4D111structure(PDBID:4YBQ,chainsCandD)asthe

searchmodel,and interpretableelectron-densitymapswereobtained.Electron

densities corresponding to either TNFα or RANKL were observed, andmodel

buildingandstructurerefinementareunderway.

Discussion

Fabfragmentsarethemostcommonlyusedcrystallizationchaperones.However,

theyarerelativelylargeco-crystallizationpartnersofapproximately500amino

acids,andtheyoftengeneratecrystallatticeswithoneormorelargecelledges,

which can cause diffraction spots to overlap and complicate structural

determination. The flexibility of the elbow angle between the variable and

constant domains in Fab fragments sometimes hinders crystallization. Fv

fragments can be used as complementary and alternative crystallization

chaperoneswithhavingallthefeaturesofthebindinginterfacepresentinlarger

Fabfragments.Single-chainFv(scFv)fragmentsaresimilartoFvfragments,but

the linker between the VL and VH domains makes scFv fragments prone to

forming intermolecularly domain-swapped dimers23. The dimer-monomer

12

equilibriumof scFv fragments can increase structuralheterogeneity; therefore,

an antibody in scFv format is less desirable for crystallization. In addition, Fv

fragmentsarelesspronetodistortionthanscFvfragmentsbecauseaggregation

of scFv fragments is sensitive to the linker length. Nanobodies (VHHs) are

increasinglyfindingfavorwithcrystallographersduetotheirsmallsize,easeof

expressionandpurification,andextremestructuralstability.Anotherfascinating

featureofNanobodiesistheirextendedconvexparatopestructurethatcanbind

deepintomolecularcleftstotrapthetargetinaparticularconformationalstate4.

However, Fv fragments have an advantage over Nanobodies in more efficient

enlargement of hydrophilic surfaces for crystal contacts because of the larger

molecularsize.

Fvfragmentsareheterodimericmoleculesconsistingoftwodomains,VL

andVH, thatassemble throughhydrophobic interactions.Eachof theVLandVH

domainscontainsanintrachaindisulfidebondforstabilization.Thesestructural

propertiesrequireasophisticatedapparatusforsecretion,folding,andassembly

aswellasanoxidizingenvironmentfordisulfidebondgeneration.B.choshinensis

iswellknownfor itscapacitytosecrete largeamountsofproteinsdirectly into

the culture medium. Expression of Fv fragments via the B. choshinensis Sec-

dependent secretionpathwayensures the formationof the intrachaindisulfide

bonds to stabilize the structure. Thus, we combined the advantages of

monocistronic stoichiometric expressionwith those of Gram-positive bacterial

secretoryexpressiontodeveloptheiRATsystem.Tomakethissystemsuitable

forstructuralstudies,thelinkersegmentthattetherstheVLandVHdomainscan

be cleaved by the site-specific protease and removed during the purification

13

process.

The iRAT system is a streamlined pipeline that provides the desired

intermediatethroughputtoproducemanyFv fragments inparallel.Thetypical

durationforrunningonecycleoftheiRAT-productionprocedure,fromplasmid

constructiontoproteinpurification,is10days.Theprocedureiseasyandcost-

effectiveanddoesnotrequireanytime-consumingorlabor-intensivesteps.The

iRAT systemgenerally produceshighFv fragmentpurification yields sufficient

for structural studies. We have tested the iRAT-based expression and

purification of randomly chosen 17 different Fv fragments and found that the

yieldsexceed3mgperliterofculturein12outofthem(datanotshown).These

yieldsconsiderablyexceedthoseobtainedfromequivalentconstructsexpressed

in theE.coli periplasm.A limitationof the iRAT system is the yield variability

amongindividualantibodyclones,whichcanbeattributedtothesequenceofthe

variabledomainsandthestabilityofthehydrophobicinteractionsbetweenthem.

However,wehavenotidentifiedspecificsequencemotifsandotherfactorsthat

affecttheyields;hencetheyieldcannotbejudgedinadvanceonthebasisofthe

sequenceinformationalone.

Overall,theiRATproductionsystemisanaccessiblemethodforuseina

broad range of Fv antibody-mediated crystallography and structure-based

research on the binding modes of therapeutic antibodies. Moreover, it may

generally be applicable to the large-scale production of antibody reagents and

therapeutics.

14

Methods

Plasmidconstruction

ThepBIM2vector(Fig.1A)forexpressionofFvfragmentswasdesignedonthe

basis of the backbone of theBrevibacillus plasmid pNY326 (Clontech). TheVL-

insertionsite,iRATcassetteandVH-insertionsiteweretandemlyarrangedinthis

orderandintroduceddownstreamoftheconstitutivelyactiveP5promoterand

theSecsecretionsignalsequence.TheiRATcassetteencodedtheN-terminalTEV

cleavagesite,His6tag,a9-residueGly/Ser-richlinker,theMBPcodingsequence

(residues 27 to 392 fromUniProt ID: P0AEX9), a 25-residue Asn/Gly/Ser-rich

linkerandaC-terminalTEVcleavagesite(SupplementaryFig.1).Residues1-107

(Kabatnumberingscheme)oftheVLdomainand1-113oftheVHdomainderived

from monoclonal antibodies were used for Fv fragment expression. The

artificiallysynthesized,codon-optimizedcDNAsencodingtheVLandVHdomains

wereamplifiedwithprimersthatincluded5’overhangs(27bp)complementing

the upstream and downstream sequences to the pBIM2 vector junctions. The

iRAT segment was amplified with the two primers BIM2-F (5’-

ACTAGTGAAAATTTATATTTTCAAGGTCACCATCACCATCACCATTCCTCTGGTGGC

GGT) and BIM2-R (5’-

AAGCTTCGATTGGAAGTACAGGTTTTCGGAAGATCTAGAGGAACCACCCCCACCCCC

GAG).ThepNY326backbonewasamplifiedwiththetwoprimerspNY326-F(5’-

GAATTCGGTACCCCGGGTTCGA) and pNY326-R (5’-

GGATCCTGCAGCGAAAGCCATGGGAGC). A total of four PCR-amplified DNA

fragments foreachantibodyclonewereadded toGibsonAssemblyMasterMix

(NewEnglandBiolabs)andincubatedat50°Cfor60min.B.choshinensisHPD31-

15

SP3 cells (Clontech) were electroporated with the assembled DNA under

conditionsof7.5kV/cm,25μF,and1000Ω.Thetransformantswereselectedon

plates supplemented with 50 mg/L neomycin. The resulting plasmids were

extractedwithaconventionalminiprepkit.ForSPRexperiments,aderivativeof

Fv4D111withanHAtagat theC-terminusof theVHdomainwasconstructed.

PreparationofthepBIM2-basedplasmidtypicallytook3days.

ConstructionoftheexpressionplasmidforHomosapiensTNFαwasalso

on the basis of the pNY326 plasmid backbone. The extracellular domain of

humanTNFα(UniProtID:P01375,residues77to233)wasFLAG-taggedatthe

N-terminus.Tofacilitatedetectionandpurification,aTEVcleavagesiteandHis6-

HAtagwereaddedattheC-terminus.Theconstructwasinserteddownstreamof

theP5promoterandSecsecretionsignalsequence.

Constructionof theexpressionplasmid forH.sapiensRANKLwasbased

onthebackboneoftheE.coliplasmidpBAD-MycHis(ThermoFisherScientific).

TheextracellulardomainofhumanRANKL(UniProtID:O14788,residues162to

317)wasfusedwithNusA,aHis6tag,a23-residuelinkerandaTEVcleavagesite

attheN-terminus.TheconstructwasinserteddownstreamofthePBADpromoter.

E. coli TOP10 cells (Thermo Fisher Scientific) were transformed with the

resultingplasmid.ThetransformantswereselectedonLBplatessupplemented

with100mg/Lampicillin.Alloftheclonedinsertswereverifiedbysequencing

bothstrands.

Proteinexpressionandpurification

16

The Fv fragments and human TNFα were expressed in B. choshinensis and

secretedintotheculturemedium.B.choshinensiscellsharboringtheexpression

plasmidsweregrownovernightat30°Cand200rpm in2SYmedium(soytone

40g/L, yeast extract5g/L, glucose20g/L, andCaCl20.15g/L) supplemented

with50mg/Lneomycin.Theculturewasdiluted1:100into1Lof2SYmedium

in 2.5 L baffled Tunair shaker flasks and grown for 48-60 h. The cells were

removedby centrifugationat6,000g for15min.The culture supernatantwas

adjusted to a final ammonium sulfate concentration of 60% saturation. The

precipitatewaspelleted,dissolved inTBSbuffer (10mMTris-HCl,pH7.5,150

mMNaCl),anddialyzedovernightagainstthesamebuffer.Thedialyzedsample

wasmixedwithNi-NTAresin(Qiagen)equilibratedwithbufferA(10mMTris-

HCl,pH7.5,150mMNaCl,and20mMimidazole).Boundproteinswereeluted

withbufferB(10mMTris-HCl,pH7.5,150mMNaCl,and250mMimidazole),

mixed with TEV-His6 and dialyzed overnight against TBS buffer. The cleaved

His6-tagged portion (the iRAT segment of the Fv-containing polyprotein or C-

terminalHis6-HAtagoftheTNFα)andTEV-His6wereremovedusingaHisTrap

HP column (GE Healthcare) equilibrated with buffer A. The flow-through

fractions were concentrated and loaded onto a HiLoad16/60 Superdex75

column (GEHealthcare) equilibratedwithTBSbuffer.Thepeak fractionswere

pooled, concentrated, flash frozen in liquid nitrogen, and stored at -80°C. The

typicaldurationforthisprocedurewas7days.

E.coli TOP10 cells harboring the expressionplasmid forhumanRANKL

weregrownovernightat37°Cand250rpminLBsupplementedwith100mg/L

ampicillin.Theculturewasdiluted1:100into200mLofthesamemediumand

17

grownat37°CtoanOD600of0.5followedbyovernightincubationat20°Cwith

0.02%arabinose.The cellswereharvestedby centrifugation at 6,000g for 15

minandre-suspended inbufferS1 (10mMTris-HCl,pH8.0,150mMNaCl,20

mM imidazole, 0.1%TritonX-100,RocheEDTA-free protease inhibitor tablet).

Cellsweredisruptedbysonication followedbyDNaseI treatmenton ice for30

min.ThelysatewasclarifiedviacentrifugationandloadedontoaHisTrapCrude

column(GEHealthcare)equilibratedwithbufferA.Boundproteinswereeluted

withbufferB,mixedwithTEV-His6 anddialyzedovernight againstTBSbuffer.

The cleaved NusA-His6 tag and TEV-His6 were removed using a HisTrap HP

column equilibrated with buffer A. The flow-through fractions were

concentratedandloadedontoaSuperdex20010/300GLcolumn(GEHealthcare)

equilibratedwithTBSbuffer.Thepeakfractionswerepooled,concentrated,flash

frozeninliquidnitrogen,andstoredat-80°C.

Rat GLUT5 was expressed and purified using previously described

methods14.

SPRexperiments

SPR experiments were performed at 20°C using a Biacore T100with series S

streptavidin sensor chips (Series S sensor chip SA; GE Healthcare) in TBS

containing0.05%dodecyl-β-D-maltopyranoside(DDM).Biotinylatedanti-HAIgG

(Rockland Immunochemicals) was captured on the streptavidin surface (flow

cells1 and2)with a400 sec injectionof50μg/mLof antibodyat10μL/min.

PurifiedsamplesoftheHA-taggedFv4D111with12mg/mLBSAand12mg/mL

18

CM-dextranwere subsequently injected (500 μL at a flow rate of 10 μL/min)

over flow cell 2. GLUT5 at various concentrations was simultaneously passed

overflowcells1and2ataflowrateof30μL/min.Associationanddissociation

times were 2 and 5 min, respectively. Regeneration consisted of a 30 sec

injection of 10 mM NaOH. Flow cell 1 was used as a reference surface. Data

analysiswasperformedusingBIAevaluationsoftware(GEHealthcare).

Thermalstabilityassay

ThethermalstabilityofGLUT5wasevaluated inthepresenceorabsenceofFv

4D111 using the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-

coumarinyl)phenyl]maleimide (CPM), as described previously24 with minor

modifications.Briefly,19.5µLGLUT5orGLUT5-Fv4D111complex(0.2nmol)in

TBS containing 0.02%DDMwasmixedwith 0.5 µL CPM dye (4mg/mL). The

reactionmixturewas transferred to a cleanPCR tube andheatedwith a ramp

rateof3°C/mininaMyiQ2thermalcycler(Bio-Rad).Theexcitationwavelength

wassetto365nmandtheemissionwavelengthwassetto460nm.Assayswere

performedoveratemperaturerangeof4°Cto80°C.Meltingtemperatures(Tm)

weredeterminedbyfittingthecurvestoasigmoidaldose-responseequation.

Crystallization

TheproteincomplexwaspreparedbyincubatingthetargetproteinwiththeFv

fragment at amolar ratioof1:2 for1hon ice. Size exclusion chromatography

wasperformedonthecomplex(Superdex20010/300column,GEHealthcare),

19

whichwasequilibratedwithTBScontaining0.02%DDM(GLUT5-Fv4D111)or

TBS (TNFα-certolizumab Fv and RANKL-denosumab Fv). Peak fractions

containing the complexes were concentrated to approximately 10 mg/mL by

ultrafiltration (Millipore, MWCO 10 kDa) and used for the crystallization

experiments.

The crystals used for the X-ray diffraction experimentswere grown for

14-20 days at 20°C by hanging drop vapor diffusion. A 400 µL reservoir was

equilibrated against a 2µL drop containing a 1:1mixture of the complex and

reservoir solution. For GLUT5-Fv 4D111 co-crystallization, the reservoir

contained0.1MTris-HCl(pH8.0),0.12MCaCl2and33-35%PEG400.ForTNFα-

certolizumab Fv co-crystallization, the reservoir contained 0.1 M tri-sodium

citrate/citric acid (pH 5.6) and 50% PEG3000. For RANKL-denosumab Fv co-

crystallization,thereservoircontained0.1Msodiumacetate/aceticacid(pH4.6)

and 2 M NaCl. The GLUT5-Fv 4D111 co-crystals were flash-frozen in liquid

nitrogen. The co-crystals of TNFα-certolizumab Fv and RANKL-denosumab Fv

werecryo-protectedin25%ethyleneglycolinthemotherliquorandthenflash-

frozeninliquidnitrogen.

Diffractiondatacollectionandanalysis

Thediffractiondatawere collected at100Kat the SPring-8beamlineBL41XU

(Japan) using a MX225HE (Rayonix) or PILATUS3 6M (Dectris) detector. The

data were then integrated and scaled using HKL200025 or XDS26. Molecular

replacement was performed with the MR-PHASER program27. The atomic

20

coordinatesofFv4D111 (PDB ID:4YBQ, chainsCandD) servedas the search

models.ThemodelwasfurtherrebuiltinCOOT28.

Acknowledgments

Thediffractiondatawere collectedat SPring-8 (ProposalNos.2015A1104and

2016A2570)withtheexcellentassistanceof thebeamlinescientists.Thiswork

was funded by the Research Acceleration Program of the Japan Science and

TechnologyAgency (JST);by thePlatform forDrugDiscovery, Informatics, and

StructuralLifeScienceoftheMinistryofEducation,Culture,Sports,Scienceand

Technology(MEXT)ofJapan;andbyGrants-in-AidsforScientificResearchfrom

the Japan Society for the Promotion of Science (JSPS) (Nos. 15K06968 and

15J04343).S.H.isarecipientofaJSPSpostdoctoralfellowship.

SupplementaryMaterialFilenames

SupplementaryFigure1(Filename:iRAT_FigS1.tif)

ConflictsofInterestStatement

Theauthorsdeclarenoconflictofinterestassociatedwiththismanuscript.

21

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Table1.Datacollectionstatistics

TNFα-certolizumabFv

complex

RANKL-denosumabFv

complex

X-raysource BL41XU,SPring-8 BL41XU,SPring-8

Wavelength(Å) 1.0 1.0

Spacegroup C2 I4132

Celldimension

a,b,c(Å) 101.4,110.45,133.6 192.5,192.5,192.5

α,β, γ (°) 90.0,105.3,90.0 90.0,90.0,90.0

TotalNo.ofreflection

measured

496486(21314) 1128891(80799)

No.ofunique

reflections

82561(6027) 29117(2123)

Resolutionrange(Å) 50-2.10(2.15-2.10)

48.15-2.25(2.31-2.25)

Rmerge 11.0(90.2) 20.6(519.5)

MeanI/σ(I) 10.3(1.97) 17.3(1.40)

Completeness(%) 99.7(98.8) 100.0(100.0)

CC1/2 99.6(52.9) 99.9(50.4)

Redundancy 6.0(3.5) 38.8(13.7)

VM(Å3Da-1) 2.78 3.50

Solventcontent(%) 55.8 64.9

Valuesinparenthesesarefortheoutershell.

27

Figure1. ExpressionconstructforproductionofFvfragmentsviaiRATsystem.(A)Schematic

diagram and construction strategy of the pBIM2 plasmid. The VL- and VH-encoding cDNAs are

amplifiedwithPCRprimerscontaining27-nucleotides5’overhangs,whichactas ‘guidingends’

inthesubsequentGibsonassemblyreaction.(B)iRAT-systheticpolyproteinsareusedtoproduce

Fv fragments in high quality and quantitywithBrevibacillus secretory expression system. The

resultingpolyproteinsarerecoveredformtheculturesupernanant,purifiedbyIMACandexcised

withTEV-His6(indicatedby ‘scissors’ icon).Afterremovalofthe iRATportionandTEV-His6by

reverseIMAC,non-taggedFvfragmentsarefurtherpurifiedbySEC.VLiscoloredinred,iRATin

greenandVHinblue.VLN-terminus,VHC-terminusandCDRsurfacearemarked.

28

Figure 2. iRAT-basedproductionandcharacterizationof theFv4D111. (A)Theconcentrated

final product frompurification, analyzedby SECon a Superdex7510/300GL column. (B) SDS-

PAGEanalysis of thepeak fractionof SEC in (A). (C) SECprofile for the isolationofGLUT5-Fv

4D111complexinthepresenceofanexcessofFv4D111(blueline)onaSuperdex20010/300GL

column. As a reference, GLUT5 alone was separated on the same column (red line). Elution

volumesofproteinstandardsareindicatedatthetop.Peak1:GLUT5-Fv4D111complex,peak2:

freeGLUT5,peak3:freeFv4D111.(D)SDS-PAGEanalysisofthecorrespondingpeakfractionsin

(C). (E) SPR sensorgrams for the association of GLUT5 (45, 90, 180 nM)with immobilized Fv

4D111werefittedto1:1Langmuirbindingmodel,givingtheapparentdissociationconstant(KD),

association rate constant (kon) and dissociation rate constant (koff) of complex formation. (F)

EffectofFv4D111onthermalstabilityofGLUT5evaluatedbyCPMassay.GLUT5alonewasused

as the control experiment (red line) compared to GLUT5-Fv 4D111 complex (blue line). (G)

RepresentativeX-raydiffractionofacrystaloftheGLUT5-Fv4D111complex.Anexampleofthe

complexcrystalsisshownintheinset.

29

Figure3. Fvfragmentsderivedfromthetherapeuticantibodies,certolizumabanddenosumab,

producedvia iRATsystem. (A)ASECchromatogramfor the isolationofTNFα-certolizumabFv

complex in thepresenceof anexcessof theFvonaSuperdex20010/300GLcolumn.PeakTC:

TNFα-certolizumab Fv complex, peak C: free certolizumab Fv. (B) SDS-PAGE analysis of the

corresponding peak fractions in (A). (C) Crystals of the TNFα-certolizumab Fv complex. (D) A

SEC chromatogram for the isolation of RANKL-denosumab Fv complex in the presence of an

excessoftheFvonaSuperdex20010/300GLcolumn.PeakRD:RANKL-denosumabFvcomplex,

peakD:freedenosumabFv.(E)SDS-PAGEanalysisofthecorrespondingpeakfractionsin(D).(F)

CrystalsoftheRANKL-denosumabFvcomplex.

30

Supplementary Figure 1. Structure of the iRAT-based Fv expression vector pBIM2. (A)

Schematic illustration of the region containing the promoter, secretion signal and iRAT-

polyproteinconstruct.(B)Sequencecorrespondingtotheregionshownin(A).P5-35andP5-10

representthe-35and-10boxofthepromoterP5,respectively.SD1andSD2areShine-Dalgarno

sequences.TheSecsecretionsignal,TEVrecognitionsequence,His6 tagandMBPareshown in

gray, orange, yellowandgreen, respectively.Grayarrowheads indicate the signal cleavage site

andTEVcleavagesites.