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bioRxiv preprint first posted online Jan. 30, 2017; doi: http://dx.doi.org/10.1101/104455. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. Expressing Biologically Active Membrane Proteins in a Cell-Free Transcription-TranslationPlatform ShaobinGuoa,*,AmitVaishb,QingChenb,*andRichardM.Murrayc a BiochemistryandMolecularBiophysics,CaliforniaInstituteofTechnology,California,USA b DiscoveryAttributeSciences,AmgenInc.,ThousandOaks,California,USA c ControlandDynamicalSystems,CaliforniaInstituteofTechnology,California,USA * Correspondingauthor:[email protected],[email protected] Abstract: Cell-freetranscription-translationplatformshavebeenwidelyutilizedtoexpresssolubleproteins inbasicsyntheticbiologicalcircuitprototyping.Fromasyntheticbiologypointofview,itiscritical to express membrane proteins in cell-free transcription-translation systems, and use them directly in biocircuits, considering the fact that histidine kinases, G-protein coupled receptors (GPCRs) and other important biosensors are all membrane proteins. Previous studies have expressed membrane proteins in cell-free systems with the help of detergents, liposomes or nanodiscs,buthavenotdemonstratedtheabilitytoprototypecircuitbehaviorforthepurpose oftestingmorecomplexcircuitfunctionsinvolvingmembrane-boundproteins.Builtonprevious efforts,inthisworkwedemonstratedthatwecouldco-translationallyexpresssolubilizedand activemembraneproteinsinourcell-freeTX-TLplatformwithmembrane-likematerials.Wefirst tested the expression of several constructs with β1 and β2 adrenergic receptors in TX-TL and observedsignificantinsolublemembraneproteinproduction.Theadditionofnanodiscstothe cellfreeexpressionsystemenabledsolubilizationofmembraneproteins.Nanodiscislipoproteinbased membrane-like material. The activity of β2 adrenergic receptor was tested with both fluorescence and Surface Plasmon Resonance (SPR) binding assays by monitoring the specific bindingresponseofsmall-moleculebinders,carazololandnorepinephrine.Ourresultssuggest thatitispromisingtousecell-freeexpressionsystemstoprototypesyntheticbiocircuitsinvolving singlechainmembraneproteinswithoutextraprocedures.Thisdatamadeusonestepcloserto testingcomplexmembraneproteincircuitsincell-freeenvironment. Introduction: Cell-free transcription-translation systems have shown to be extremely useful in synthetic biologicalcircuitprototyping[1,2].Typicalcell-freetranscription-translationsystemsarebased onS30E.coliextract[3]andtherehavebeenmanydifferentversions.Thespecificversionused inthispaperreferredtoas“TX-TL”,hasbeenoptimizedforprototypingsyntheticbiocircuits[4, 5].Unlikeothercell-freeproteinexpressionsystems,includingthePUREsystem,whicharebased onbacteriophagetranscriptionbysupplementingbacteriophageRNApolymerasestothecrude cytoplasmicextracts[6],TX-TLhasbeenshowntobesuitableforcomplexbiochemicalsystems [1,2,4,7]. Membraneproteinsplayimportantrolesintheproperfunctioningofcellsandorganisms.They laythefoundationformanybiosensorsandsignalpathwaysincells[8].Somemembraneproteins actasionchannelstotransportionsacrossmembranes[9];othersmakeuptheessentialparts bioRxiv preprint first posted online Jan. 30, 2017; doi: http://dx.doi.org/10.1101/104455. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. ofsensorysystemwhichareresponsibleforcellcommunication[10],whilemembraneenzymes catalyzeimportantchemicalreactionsnearmembranes[11].Inaddition,membraneproteinsare themostimportantdrugtargets,forexamplemorethanhalfoftherapeuticsfortreatmentof various modalities ranging from cancer to cardiovascular diseases target membrane proteins [12]. Membrane proteins are difficult targets as compared to soluble proteins because of the challengesassociatedwiththeirexpression,solubilization,andstabilization.Typicalstudiesof membraneproteinsrelyonproteinsproducedfromcellsandsolubilizedcellmembraneusing detergents, liposomes or other membrane-like materials. These approaches may have advantagesintermsofproteinyield,theyarenotwell-suitedforhigh-throughputselectionof protein constructs, since transformation, cell growth, lysis, membrane solubilization and purificationareinvolvedforeachconstruct[13].Itisalsodifficulttousethesetechniquesfor biocircuitsprototyping,whichrequirepromptfunctionofthenewlyexpressedproteins. Recently cell free expression of membrane protein have been reported using detergents, liposomesornanodiscstosolubilizeandstabilizetheproteins[13-19].Ourdataindicatedthe possibilitytointegratemembraneproteinsdirectlyintofuturesyntheticbiocircuitswithcomplex functions.Amongalltheimportantmembraneproteins,wepickedGproteincoupledreceptors (GPCRs), beta-2(and 1) adrenergic receptors (β2AR/β1AR) as model proteins. With more than 900members,GPCRsareoneofthemostimportantandthelargestintegralmembraneproteins familyinhumancellsandthemostimportantclinicaldrugtargetsastheyplayimportantrolesin manyphysiologicalfunctionsandimplicatedinmanydiseases[12,20,21].GPCRsallsharethe sametopology–seventransmembranea-helices,andtheyarethoughttofunctioninmonomeric form[22],althoughtherehavebeenstudiesindicatingtheirdimerization[23,24].WeusedTXTL platform in combination with nanodisc for expressing β2AR/β1AR and subsequent in situ stabilization.Furthermore,biologicalactivityoftheseproteinswasconfirmedbybindingtoits ligands. bioRxiv preprint first posted online Jan. 30, 2017; doi: http://dx.doi.org/10.1101/104455. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. A B Figure1.IllustrationoftheplasmidmapandlinearDNAoftheconstructs.pSG73isusedasanexample.pSG74and pSG75sharethesamefeaturesaspSG73.A:CircularplasmidmapofpSG73.B:LinearDNAversionofpSG73. ResultsandDiscussion: We ordered gene synthesis services and then used golden gate assembly [27] to make three differentGPCRconstructswiththreedifferentvariants,whichsharesimilarbackbone,promoter, ribosomebindingsitesandterminator(Figure1);Figure2liststhedifferenceincodingsequences correspondingtovariousadrenergicreceptorconstructs.Allthreeconstructssharedthesame superfolder GFP (sfGFP) fusion protein topology, which was tagged with 6xHis tag at the Cterminaloftheprotein.sfGFPisusedformonitoringandestimationoftargetproteinexpression levelduringandattheendofTX-TL.Whereas,6xHistagwasusedtodetectproteinsinWestern, tocapturetheproteinforbindingassays,andforaffinitypurification,ifnecessary. OneoftheadvantagesofTX-TLexpressionplatformisthatwecoulduseeitherlinearDNAor plasmid DNA for expression in TX-TL [7]. Because of that, we could implement fast construct prototypinginTX-TLbyligatingpartstogetherandamplifyingthelinearDNAswithPCR,avoiding cloningthelinearfragmentsintoplasmid.ToexpresstheseconstructsinTX-TL,weonlyadded GPCR%Protein Construct Protein%Size β1#AR#ts:)3ZPR)Thermostabilized)turkey)β1)adrenergic)receptor pSG73:)protein#sfGFP#His6 65kD)(35kD)minus)sfGFP) β2#AR)wild)type:)ADRB2_HUMAN)β2)adrenergic)receptor pSG74:)protein#sfGFP#His6 75kD)(45kD)minus)sfGFP) β2#AR#T4L:)2RH1)β2#adrenergic)receptor/T4#lysozyme)chimera pSG75:)protein#sfGFP#His6 85kD)(55kD)minus)sfGFP) Figure2.Informationof GPCR constructs usedinexperiments.pSG73andpSG75havethecorrespondingPDBnumber noted.Proteinsizeofeachfusionproteinisalsolisted. bioRxiv preprint first posted online Jan. 30, 2017; doi: http://dx.doi.org/10.1101/104455. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. DNAoftheseconstructstoTX-TLreactionmix.Theiterationofeachprototypingtestsignificantly faster(overnight)comparedtoweeksusingcloningforcell(E.coli)expression. A B 300 1200 1000 GFP-expression- in-nM GFP+expression+ in+nM 250 200 150 100 50 800 600 400 200 0 0 pSG73:+ b1ARts+ linear+ 10nM pSG73:+ b1ARts+ linear+ 20nM pSG73:+ b1ARts+ linear+ 40nM pSG74:+ b2AR+ linear+ 10nM pSG74:+ b2AR+ linear+ 20nM pSG74:+ pSG75:+ pSG75:+ pSG75:+ b2AR+ b2ART4L+ b2ART4L+ b2ART4L+ linear+ linear+ linear+ linear+ 40nM 10nM 20nM 40nM pSG73:b1ARtslinear20nM pSG73:- pSG73:- pSG74:- pSG74:- pSG74:- pSG75:- pSG75:- pSG75:b1ARts- b1ARtsb2ARb2ARb2AR- b2ART4L- b2ART4L- b2ART4Llinear- w/- linear- w/- linear- linear- -w/- linear- w/- linear- linear- w/- linear- w/ND1 ND3 20nM ND1 ND3 20nM ND1 ND3 Figure3.ExpressionlevelofpSG73-75constructsinTXTLmeasuredbysfGFPA:EndpointmeasurementofGFPexpression from10nM,20nM and40nMlinearDNAofpSG73,pSG74 andpSG75 in10µLTX-TL.B: Endpoint measurementof GFP expressionfrom20nMlinearDNAofpSG73-75withorwithout24 µMnanodiscsinTX-TL.ND1isMSP1D1-DMPCandND3is MSP1E3D1-DMPG.Measurements weredoneat29°C in BIOTEKSynergyH1HybridMulti-ModeMicroplateReaderusing ex485nm/em525nm. WefirstestimatedtheexpressionlevelofpSG73-75inTX-TLbymeasuringthefluorescenceof sfGFP which was fused at the C-terminal of β2AR or 1AR, using a plate reader at 485 nm (absorbance)/525nm(emission).AllthelinearDNAconstructsshowedaGFPfluorescencesignal, indicatingsuccessfulexpressionofthefusionproteinsinTX-TL(Figure3A).Differentconstructs, despitehavingexactlythesamepromoter,ribosomebindingsite,fusionproteinframeworkand DNA concentration, showed different expression levels of sfGFP, especially pSG74. This suggestedthatthedifferenceincodingsequencescouldaffecttranscriptionandtranslationlevel. AnotherobservationwasthatincreasinglinearDNAconcentrationcouldhelpincreasethefusion proteinexpression,butthisincreasewaslimitedbyTX-TLresourcesand/ortoxicsaccumulated inbatchmode,asshownelsewhere[28].SettingupTX-TLreactionsindialysissystemsislikelyto improvetheproteinexpressionlevel. After testing the fusion protein expression level, we explored the possibility of stabilizing hydrophobicmembraneproteinbyprovidingamembranemimicintothereaction.Detergent micelle,liposomeandnanodiscarecommonlyusedtoprovideartificialbilayerenvironmentto membraneprotein(Ref).WeobservedthatmajorityofdetergentsweredetrimentaltoTX-TL reactioncomprisingmembraneprotein(datanotshown).Conversely,reconstitutingproteininto liposome resulted into a poor yield of the folded protein, which could be attributed to the liposome’s closed topography. Therefore, we employed nanodisc − a lipid-protein complex, composed of lipids constrained by a membrane scaffold protein (MSP). Nanodisc provides a robustplatformwithtwo-dimensionaltopographyforreconstitutingTX-TLexpressedmembrane protein.Wechosetwoofthemostcommonlyusednanodiscs:MSP1D1-DMPC(ND1)~10nm diameterandalongerversion,MSP1E3D1-DMPG(ND3)~13nmindiameter[13]. bioRxiv preprint first posted online Jan. 30, 2017; doi: http://dx.doi.org/10.1101/104455. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. A No*DNA pSG73:*β14AR4ts 10nM 20nM 40nM pSG74:*β24AR 10nM 20nM 40nM pSG75:*β24AR4T4L 10nM 20nM 40nM Dimer Monomer B pSG73:*β14AR4ts Nanodisc ! ND1%%%%%ND3 pSG74:*β24AR ! ND1%%%%%ND3 pSG75:*β24AR4T4L ! ND1%%%%%ND3 Dimer Monomer Figure4.WesternblotsofTX-TLreactions.A:ResultsofwholereactionTX-TLsamples.Aftermeasuringthefluorescence oftheTX-TLreaction,theywereusedforwesternblotusingthemethodinMaterialsandMethods.TX-TLreactionwith noDNAwasusedasnegativecontrolandthreedifferentconcentrationsofthreedifferentconstructswererununderthe same condition. B: Results of only supernatant from TX-TL samples. Only soluble samples were run on this blot. SupernatantswithoutanynanodiscswererunatthesameconditionasoneswitheitherND1orND3. WerepeatedtheTX-TLexpressionexperimentinthepresence/absenceofND1orND3.Asshown inFigure3B,thepresenceofnanodiscimprovestheTX-TLefficiency,asindicatedbyenhanced GFP fluorescence. Nanodisc doesn’t have any intrinsic fluorescence, therefore the increased fluorescenceshouldbearisingfromthefusionprotein.GPCR-sfGFPfusionproteinprecipitated in the TXTL reaction mix without nanodisc. However, in the presence of nanodisc, the supernatantofthereactionmixtureexhibitedincreasedGFPfluorescencesignal,indicatingthat nanodiscslikelytohelpstabilizingfusionmembraneproteinsinTX-TL,andkeepthefoldedfusion proteininsupernatant. TofurtherconfirmthatthetargetfusionproteinswereexpressedinTX-TLandnanodiscscould helpsolubilizeGPCR-sfGFPfusionproteins,weranSDS-PAGEofTX-TLsamplesandwesternblots withananti-His6antibody.First,weranthewholereactionsamplesofthreedifferentconstructs atthreedifferentconcentrations(Figure4A). As illustrated on the blot, samples that had one of the three constructs showed significant detection of His-tagged proteins, protein size was confirmed on the blot using a ladder. In contrast,controlsamplewithwithoutDNAhadnoHis-taggedsignal.Additionally,therewerenot significantdifferencesbetweendifferentconcentrationsinpSG73andpSG75,suggestingmasstransfernutrientslimitationandtoxicaccumulationintheTX-TLreactionasdiscussedearlier. AnotherinterestingobservationwasthatpSG73-β1-AR-tsdidnotshowdimerizationbutpSG74 bioRxiv preprint first posted online Jan. 30, 2017; doi: http://dx.doi.org/10.1101/104455. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. andpSG75,whichwerebothβ2-ARbasedfusionproteins,showedstrongdimerbandsonthe blot.ThepresenceofGPCRdimerizationisconsistentwithpreviousreports[24]. We further tested the presence of ND by spinning down the TX-TL reaction mix and took supernatantonlytorunthewesternblot.Alltheinsolubleproteinswouldprecipitateoutand endedupinthepelletaftercentrifugation.Onlysolubleproteinswouldbeinthesupernatant andcanbedetectedinthewesternblot.InFigure4B,wehadthreedifferentconstructsandeach ofthemhadthreedifferentexperimentalconditions:nonanodisc;withND1:MSP1D1-DMPC; and with ND3: MSP1E3D1-DMPG. All target proteins generated in TX-TL precipitated without nanodiscsandleftinthepellet(confirmedbyrunningpelletonwesternblot,datanotshown). Onthecontrary,wheneitherND1orND3wasaddedintothereaction,wesawsignificantbands of target membrane proteins by the Western, suggesting that these hydrophobic membrane proteinsbecamesolublewithhelpfromnanodiscs. SofarwehavedemonstratedthattargetmembraneproteinsproducedinTXTLreactionscanbe solubilizedandstabilizedwithin-situpresenceofnanodiscs.However,proteinassociationwith nanodiscdoesn’tinsurethattheseproteinsarebiologicallyactive.Tofurthertestwhetherthese soluble proteins were active, we designed two binding assays: fluorescence-based carazolol bindingassayandSPRbasednorepinephrinebindingassay. Althoughthereisnoneedtopurifytheproteinforcircuitprototyping,purificationisrequiredto confirmthebindingactivityofTX-TLexpressedproteintoavoidinterferencecausedbyE.coli endogenousproteinsintheTX-TLreactionmix. We used Ni-NTA affinity chromatography to purify His-tagged target protein. The protocol is describedinMaterialsandMethods. Fluorescence-based carazolol binding assay uses (S)-carazolol, a derivative of the potent β blockercarazololwithfluorescenceproperties(ex633nm/em650nm).Thisligandcanbeusedas afluorescencetrackerforβ2ARbindingactivity.Thedetailedexperimentalsetupisdescribedin Materials and Methods. Briefly, purified membrane proteins were incubated with or without carazolol for one hour. These samples were further dialyzed against 100X volume buffer overnight.Subsequently,allthesampleswereconcentrateddowntotheirstartingvolumeand fluorescence signal was measured in plate reader. Green fluorescence (GFP) indicates the amountoffusionproteinsinthesamplesandredfluorescencewouldrepresenttheamountof carazololboundtotargetmembraneproteinsasanindicationofproteinactivity. WeusedtwodifferentND1(MSP1D1-DMPC)inthisassay:1)HisNDuses6xHistaggedMSP1D1 andwaspurchasedfromCubeBiotech;2)BiotinNDusesbiotinylatedMSP1D1andwasmadein house.AsseeninFigure5B,therewasnotmuchdifferenceingreenfluorescencesignalbetween the samples. However, there is a significant difference between ND1 samples incubated with bioRxiv preprint first posted online Jan. 30, 2017; doi: http://dx.doi.org/10.1101/104455. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. carazolol. We attribute high carazolol signal on HisND reconstituted β2AR to the presence of emptyNDafterNi-NTAaffinitychromatographypurification. One limitation of fluorescence-based assay is that non-specific binding of carazolol to lipids surroundingmembraneproteincouldcausehighersignal-to-noiseratio.Toovercomethis,we developedaSPR-basedbindingassayusingnorepinephrine,whichisapartialagonistforβ2AR. Since norepinephrine shares the same binding pocket with carazolol [31], we tethered norepinephrinetothesurfaceviaamidebondbetweenitsprimaryamineandthecarboxylgroup on the surface. We hypothesized that carazolol could be used as a competitor for β2AR to norepinephrinebindingonSPR.AsshowninFigure5C.,SPRbindingresponseofβ2ARdecreased ~60%whenitwasincubatedwith1µMcarazolol,indicatingspecificinteractionbetweenβ2AR anditsbindingpartners. A B 20 1.2 1 16 GFP+fluorescence+fold+change Carazolol+fluorescence+fold+change 18 14 12 10 8 6 4 0.8 0.6 0.4 0.2 2 0 C 0 D 140 B in d in g 0R e s p o n s e 0o f 0N D 8 R e c o n s t it u t e d 120 b2AR Response+Unit 100 80 b2AR_car 60 40 S P R 0R e s p o n s e 0( R U ) B 2 A R 0( W T ) 0o n 0N E 8 F u n c t io n a liz e d 00S u r f a c e s 100 50 0 A 2 B A R _ C a R r 20 B 2 0 0 200 400 600 Time+(s) 800 1000 1200 Figure5.BindingassaysofTX-TLmadeβ1andβ2adrenergicreceptor.A:Barchartofcarazololfluorescencefoldchange. Thenumberwastheratioofredfluorescencesignalfromsamplesincubatedwithcarazololtotheoneswithoutcarazolol. DetailedexperimentalmethodisinMaterialsandMethods.Allsamplesweredialyzedagainstbufferwithoutcarazololto removenon-bindingcarazolol.B:BarchartofGFPfluorescencefoldchangewiththesamesamplesfromA.Thenumberwas theratioof greenfluorescence signalfrom samplesincubated with carazolol to theones withoutcarazolol. C: Response curvesofβ2adrenergicreceptorproteinsbindingtoSPRsurfacecoatedwithnorepinephrine.Redcurvewasresponsesignal fromβ2-AR sample withoutcarazolol. Green curve wasresponse signalfromsample incubated with1 µM carazolol asa control.D:BarchartoftheendpointoftheresponsecurvesinC. bioRxiv preprint first posted online Jan. 30, 2017; doi: http://dx.doi.org/10.1101/104455. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. Tosummarize,wehavedemonstratedthat1)membraneproteincanbeexpressedinTX-TLat analytical scale; 2) presence of nanodisc during TX-TL reaction facilitates folding and solubilizationofsinglechainmembraneprotein;3)fluorescenceandSPRbindingassayswere developedtodemonstratespecificinteractionbetweensmall-moleculeandnanodisc-stabilized membraneprotein. Conclusions: Inthiswork,wetestedproteinsfromtheGPCRfamilyinourcell-freetranscription-translation (TX-TL) system, which would be ideal for synthetic biocircuit prototyping. We expressed β1AR/β2AR in TX-TL with nanodiscs and were able to show that not only these membrane proteins are soluble in TX-TL with nanodiscs, but they were also active. Nanodiscs are cotranslationally associated with membrane proteins without extra processing, which enables directprototypingofacompletebiocircuitwithTX-TLproducedmembraneprotein(s). Weintendtooptimizeourbindingassaysandperformcompetitivebindingexperimentstotest the stringency of this assay. We envision that GPCR co-expressed with G protein can be expressedbyTX-TLtotestthebiologicalcircuitincludingsignaltransduction. Additionally, histidine kinases, which phosphorylate corresponding response regulators, can activatedownstreamtranscriptionandtranslation.Wehavestartedtestedsomehybridhistidine kinases and have seen promising results. Our goal is to prototype a logic biocircuit with membraneenzymeinit,expandingTX-TLplatformtobroadertopics. bioRxiv preprint first posted online Jan. 30, 2017; doi: http://dx.doi.org/10.1101/104455. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. MaterialsandMethods: PlasmidsandlinearDNAs: DNA and oligonucleotides primers were ordered from Integrated DNA Technologies (IDT, Coralville,Iowa).PlasmidsinthisstudyweredesignedinGeneious8(Biomatters,Ltd.)andwere madeusingstandardgoldengateassembly(GGA)protocols.BsaI-HF(R3535S)enzymeusedin GGAwaspurchasefromNewEnglandBiolabs(NEB).LinearDNAsweremadebyPCRingprotein expressionrelatedsequencesoutofGGAconstructsusingPhusionHotStartFlex2XMasterMix (M0536L)fromNEB. TX-TLreactions: TX-TLreactionmixwassetupaccordingtopreviousJOVEpaper[5].Briefly,TX-TLextractand bufferweremixedtogetherwithcalculatedlinearDNAsorplasmidswithorwithoutnanodiscs. Reactionvolumesvariedfrom10µL(initialscreening)to1mL(proteinpurificationandanalysis). Gelandwesternblot: GelsusedinthisworkwereBolt4–12%Bis-TrisPlusGelsfromThermoFisherScientific.Running bufferwasBoltMESSDSRunningBuffer.Gelswererunwithoutreducingagents.Proteinsamples weremixedwithLDSsamplebufferbeforeloadingintogels.iBlot2GelTransferDeviceand iBlot Nitrocellulose Regular Stacks were used for transfer proteins from gel to membrane. MembranewasthentransferredtoiBinddeviceandincubatedwithPenta-HisHRPConjugatein 1:500dilutions.BlotsweredetectedusingSuperSignalChemiluminescentHRPSubstratesfrom ThermoFisherScientific. Proteinpurification: TX-TLreactionmixwasfirstspun@14,000gfor10minat4°C.Supernatantwasthentransferred to buffer-equilibrated HisPur Ni-NTA Spin Purification column (ThermoFisher Scientific) and incubatedwithshakingfor1h.Thenspundowntheflowthrough@2000gfor2minandwashed withnanodiscbuffer(20mMTrispH7.4,0.1MNaCl)addedwith20mMimidazolethreetimes. Elutionwasdonebyaddingelutionbuffer(20mMTrispH7.4,0.1MNaCl,250mMimidazole)for 3times2columnvolume.ProteinswerethenconcentratedusingAmiconUltraCentrifugalFilter Units(Millipore)withUltracel-30membrane. Fluorescence-basedcarazololbindingassay: Carazolol was purchased from Abcam (S)-Carazolol Fluorescent ligand (Red) ab118171. Each purified protein was first divided into two equal volume samples. One was added 100nM carazolol(dissolvedinwater)andtheotheronewasaddedthesamevolumeofwater.Theywere incubated at 4°C for 1h before transferred to mini 10k MW D-Tube Dialyzers (Millipore) and dialyzed against 100x volume of nanodisc buffer for overnight. Then dialyzed samples were concentratedusingAmiconUltraCentrifugalFilterUnits(Millipore)withUltracel-30membrane to starting volume. Samples were then put into BIOTEK Synergy H1 Hybrid Multi-Mode MicroplateReaderandmeasuredforGFPfluorescence(ex485nm/em525nm)withgain61orgain bioRxiv preprint first posted online Jan. 30, 2017; doi: http://dx.doi.org/10.1101/104455. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 100orRedfluorescence(ex633nm/650nm)withoptimalgain.GFPfluorescencewasconverted tonMusingcalibrationdatafrompurifiedGFPprotein. SurfacePlasmonResonance(SPR)basednorepinephrinebindingassay: GEBiacoreT-200SPRsystemwasusedforSPRexperiment.Goldplatedchipwasfirstimmobilized withnorepinephrineandthenwashedawayextrachemical.Therearefourchannelsononechip. Twowereusedasexperimentalchannelsandtheremainingtwowereusedasnegativecontrols toprovidebackgroundresponsefrombuffer.Onesamplewasincubatedwith1µMcarazololfor 1hattotestbindingspecificityandtheothersamplewasincubatedwithsamevolumeofwater. Samples were then loaded and flowed through corresponding experimental channels and responsecurveswererecorded. Acknowledgments WewouldliketothankHaoChenforreceptorplasmiddesign.MarieWrightandRobertKurzeja fornanodiscexpression,andAmgenInc.forfinancialsupport. Reference: 1. Takahashi,M.K.,etal.,Characterizingandprototypinggeneticnetworkswithcell-free transcription-translationreactions.Methods,2015.86:p.60-72. 2. Niederholtmeyer,H.,etal.,Rapidcell-freeforwardengineeringofnovelgeneticring oscillators.Elife,2015.4:p.e09771. 3. Nirenberg,M.W.andJ.H.Matthaei,Thedependenceofcell-freeproteinsynthesisinE. coliuponnaturallyoccurringorsyntheticpolyribonucleotides.ProcNatlAcadSciUSA, 1961.47:p.1588-602. 4. Shin,J.andV.Noireaux,AnE.colicell-freeexpressiontoolbox:applicationtosynthetic genecircuitsandartificialcells.ACSSynthBiol,2012.1(1):p.29-41. 5. Sun,Z.Z.,etal.,ProtocolsforimplementinganEscherichiacolibasedTX-TLcell-free expressionsystemforsyntheticbiology.JVisExp,2013(79):p.e50762. 6. 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