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重组FLAG标签的融合表达纯化及亲和常数的测定.docx

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重组FLAG标签的融合表达纯化及亲和常数的测定 INTRODUCTION FLAGepitopetagsarecommonlyusedtofacilitatethepurificationanddetectionofrecombinantproteins.Thesetagsconsistofashortaminoacidsequence(DYKDDDDK)thatcanbefusedtotheN-orC-terminusofaproteinofinterest.TheFLAGtagisimmunoreactive,meaningthatitcanbespecificallyrecognizedandboundbyanantibody.TheFLAGtagcanbeusedforavarietyofapplications,includingWesternblotting,immunoprecipitation,andaffinitypurification. OneoftheadvantagesoftheFLAGtagisthatitcanbeeasilyremovedfromtherecombinantproteinusingaspecificprotease,suchasenterokinase.Thisallowsforthegenerationofaproteinthatisfreeofanyadditionalsequencesthatcouldaffectitsactivityorfunction. Inthispaper,wewilldescribetherecombinantexpressionofaproteinofinterestwithaFLAGtag,followedbyitspurificationusinganaffinityresin.Wewillalsodescribethedeterminationofthedissociationconstant(Kd)oftheinteractionbetweentheFLAG-taggedproteinandtheFLAGantibody. METHODS RecombinantProteinExpressionandPurification TheproteinofinterestwasclonedintoaplasmidcontainingaFLAGtagsequenceatitsC-terminus.TheplasmidwastransformedintoEscherichiacoliBL21(DE3)cells,andproteinexpressionwasinducedusingIPTG.Thecellswereharvestedandlysedusingasonicator,andthelysatewasclarifiedbycentrifugation. Thesolublelysatewasloadedontoacolumncontaininganti-FLAGresin(Sigma-Aldrich)thathadbeenequilibratedwithbindingbuffer(20mMTris-HClpH8.0,150mMNaCl).Thecolumnwaswashedwithbindingbuffertoremoveanyunboundproteins,andtheFLAG-taggedproteinwaselutedusingelutionbuffer(bindingbuffercontaining100μg/mLFLAGpeptide). TheelutedproteinwasanalyzedbySDS-PAGEandWesternblottingusingananti-FLAGantibodytoconfirmitsidentityandpurity. DeterminationofAffinityConstant Theaffinityconstant(Kd)oftheinteractionbetweentheFLAG-taggedproteinandtheanti-FLAGantibodywasdeterminedusingsurfaceplasmonresonance(SPR)withaBiacoreT200instrument(GEHealthcare). Theanti-FLAGantibodywasimmobilizedonaCM5sensorchipusingamine-couplingchemistry.TheFLAG-taggedproteinwasinjectedoverthechipatvariousco