Click chemistry based fluorescent labelling of surface displayed proteins on Escherichia coli

Nickelsen A, Nienberg C, Jose J

Abstract

Intoday'spharmaceuticalresearchproteinproteininteractions(PPI)playanimportantroleforthecharacterizationofdrugtargetsandfortheidentificationofnewtherapeutics.MostapplicationsofPPIarebasedonfluorescencedetectionrequiringafluorophorelabelledtargetprotein.Comparedtoothercommonusedlabellingstrategies,anincorporationofanunnaturalaminoacidfollowedbyaclickchemistrybasedbiorthogonalreactionwithafluorophoreisdistinguishedbyhighsite-specificity.Autodisplayisasurfacedisplaytechnologybasedonthesecretionmechanismofautotransporterproteinsingram-negativebacteriaandenablesthepresentationoftargetproteinsontheiroutermembrane[1].InthisstudyclickchemistryandAutodisplaywerecombinedtofluorescentlymodifyCK2βasamodelproteinonthesurfaceofEscherichiacoli.CK2β,theregulatorysubunitofthehumanproteinkinaseCK2,functionsasamodulatorofCK2activityandstabilityaswellasadockingplatformforCK2substrates.Wereportthesuccessfulincorporationoftheunnaturalaminoacidpara-azidophenylalanine(pAzF)[2]intothepurifiedandthesurfacedisplayedCK2β.ThereforeasuitablepositioninthesequenceofCK2βwaschosenandmutatedtotheamberstopcodon,TAG.AfterwardspAzFwasincorporatedatthispositionusinganorthogonalambersuppressortRNA.PerformingtheSPAACclickreaction(Strain-PromotedAlkyne-AzideCycloaddition)[3]withdibenzylcyclooctyne-fluorophoresthepurifiedaswellasthesurfacedisplayedCK2βwassite-specificlabelledandanalyzedbySDS-PAGE.WewereabletoverifythefluorescencemodificationofsurfacedisplayedCK2βbyflowcytometry.Withthisinnovativeprocedureofproteinlabellingonthesurfaceofbacterialcells,screeningassayandproteininteractionstudieswithwholecellswereenabled.MoreoverthismethodoffersfurtherinvestigationsontheinteractionofCK2βanditssubstratesforexamplebyfluorescenceactivatedcellsorting,fluorescenceresonanceenergytransferormicroscalethermophoresis.