pMCSG7

pMCSG7ProteinExpressionandPurification25,8–15(2021)doi:10.1006/prep.2021.1603ANewVectorforHigh-Throughput,Ligation-IndependentCloningEncodingaTobaccoEtchVirusProteaseCleavageSiteLucyStols,*MinyiGu,?LyndaDieckman,?RosemarieRaffen,?FrankR.Collart,?andMarkI.Donnelly*,1*EnvironmentalResearchDivisionand?BiosciencesDivision,ArgonneNationalLaboratory,Argonne,Illinois60439ReceivedOctober8,2021ligation-independentcloning;TEVprotease;affinityToestablishhigh-throughputmethodsforproteinpurification.crystallography,allaspectsoftheproductionandanal-ysisofproteincrystalsmustbeaccelerated.Auto-mated,plate-basedmethodsforcloning,expression,Theemergingfieldofstructuralgenomicsembracesandevaluationoftargetproteinswillhelpresearchersmanydiverseobjectives,butingeneraldependsontheinvestigatethevastnumbersofproteinsnowavailablegenerationofproteinstructuralinformationatamuchfromsequencedgenomes.Ligation-independentclon-fasterratethanoccurscurrently(1–3).Toachievethising(LIC)iswellsuitedtoroboticcloningandexpres-objective,everystepinvolvedindeterminingaprotein’ssion,butfewLICvectorsareavailablecommercially.structuremustbeaccelerated,includingthecloning,WehavedevelopedanewLICvector,pMCSG7,thatexpression,andpurificationoftargetproteins.Automa-incorporatesthetobaccoetchvirus(TEV)proteasetionoflabor-intensivesteps,whenpossible,candra-cleavagesiteintotheleadersequence.Thisproteasematicallyincreasethroughputandreducecosts(4).TheishighlyspecificandfunctionsunderawiderangeofMidwestCenterforStructuralGenomics2(MCSG,conditions.ThenewvectorincorporatesanN-terminal)hasinstitutedrobotic,microtiterhis-tagfollowedbytheTEVproteaserecognitionsiteplate-basedprotocolsforhigh-throughputcloningofandaSspIrestrictionsiteusedforLIC.Thevectortargetproteinsandanalysisoftheirexpressionandfunctionedasexpected,givinghighcloningefficienc-solubility.Theprotocolsincorporateligation-indepen-iesandstrongexpressionofproteins.Purificationanddentcloning(LIC)oftargetgenes(5,6).LICpossessescleavageofatargetproteinshowedthatthehis-tagseveraladvantagesforautomatedcloning.IteliminatesandtheTEVcleavagesitefunctionproperly.Thepro-theuseofrestrictionendonucleasedigestionandliga-teinwaspurifiedandcleavedunderdifferentcondi-tionofPCRproducts,allowinganygenetobeclonedtionstosimulatebothplate-basedscreeningmethodsintothevectorregardlessofitssequence.InLIC,PCRandlarge-scalepurificationsforcrystalproduction.primersaredesignedtoappendsequencesthat,afterThevectoralsoincludesapairofadjacent,uniquetreatmentwithT4DNApolymeraseinthepresenceofrestrictionsitesthatwillallowinsertionofaddi-asingledeoxyribonucleotidetriphosphate,generate12-tionalmodulesbetweenthehis-tagandthecleavageto15-base-pairoverhangsthatarecomplementarytositeoftheleadersequencetogenerateafamilyofvec-overhangsgeneratedinthevector.Theseoverhangstorssuitableforhigh-throughputproductionofannealsufficientlystronglytoallowthetransformationproteins.KeyWords:highthroughput;structuralgenomics;2Abbreviationsused:MCSG,MidwestCenterforStructuralGeno-mics;LIC,ligation-independentcloning;TEV,tobaccoetchvirus;1Towhomcorrespondenceshouldbeaddressed.Fax:(630)252-DTT,dithiothreitol;LB,Luria–Bertanimedium;IPTG,isopropyl7709.E-mail:mdonnelly@(Fig.1).ForpMCSG3,fromtheinitialPCRthroughtheanalysisofexpressiontheentireregionencodingthehis-tag,thrombinsite,canbeautomatedandcarriedoutinmicrotiterplates.andS-tagofpET-30Xa/LICwasexcisedbytreatmentAseriousbottleneckinhigh-throughputproteinwithNdeIandBglII.Thevectorwasdephosphorylatedstructuredeterminationisthepurificationofexpressedwithcalfintestinalphosphataseandpurifiedbyelectro-proteins.Typically,thiseffortisacceleratedbyattach-phoresisinanagarosegelandextractedusingtheQi-mentofaffinitytagstotheN-orC-terminusofex-aExgelextractionkit(Qiagen).Thesyntheticoligonu-pressedproteins(7).Sometagsalsoimprovethesolubil-cleotidesTATGCACCATCATCATCATCATTCTTCTG-ityofexpressedproteins(8)orreducetheirtoxicity(9).GTGTAandGATCTACACCAGAAGAATGATGATGA-Ideally,thesetagsarecleavedfromthepurifiedproteinTGATGGTGCA,encodingasix-histidinehis-tag,abyspecificproteasesatrecognitionsitesincorporatedsmallspacersequence,andtheappropriateoverhangs,intothe