Role of N-Terminal His6-Tags in Binding and Efficient Translocation of Polypeptides into Cells Using Anthrax Protective Antigen (PA) 英文参考文献.docVIP

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Role of N-Terminal His6-Tags in Binding and Efficient Translocation of Polypeptides into Cells Using Anthrax Protective Antigen (PA) 英文参考文献.doc

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Role of N-Terminal His6-Tags in Binding and Efficient Translocation of Polypeptides into Cells Using Anthrax Protective Antigen (PA) 英文参考文献

RoleofN-TerminalHis6-TagsinBindingandEfficient TranslocationofPolypeptidesintoCellsUsingAnthrax ProtectiveAntigen(PA) ChristophBeitzinger1.,CarolineStefani2,3.,AngelikaKronhardt1,MonicaRolando2,3,GillesFlatau2, EmmanuelLemichez2,3*,RolandBenz1,4 * 1Rudolf-Virchow-Center,DFG-ResearchCenterforExperimentalBiomedicine,UniversityofWu¨rzburg,Wu¨rzburg,Germany,2Toxinesmicrobiennesdanslarelationho?te- pathoge`nes,C3M,U1065,Inserm,Nice,France,3UFRMe′decine,IFR50,Universite′ deNice-SophiaAntipolis, Nice,France,4SchoolofEngineeringandScience,Jacobs UniversityBremen,Bremen,Germany Abstract Itisofinteresttodefinebacterialtoxinbiochemicalpropertiestousethemasmolecular-syringedevicesinordertodeliver enzymatic activities into host cells. Binary toxins of the AB7/8-type are among the most potent and specialized bacterial proteintoxins.TheBsubunitsoligomerizetoformaporethatbindswithhighaffinityhostcellreceptorsandtheenzymatic Asubunit.ThisallowstheendocytosisofthecomplexandsubsequentinjectionoftheAsubunitintothecytosolofthehost cells.HerewereportthattheadditionofanN-terminalHis6-tagtodifferentproteinsincreasedtheirbindingaffinitytothe protectiveantigen(PA)PA63-channels,irrespectiveiftheyarerelated(C2I)orunrelated(gpJ,EDIN)totheAB7/8-familyof toxins.His6-EDINexhibitedvoltage-dependentincreaseofthestabilityconstantforbindingbyafactorofabout25when thetrans-sidecorrespondingtothecellinteriorwassetto270mV.Surprisingly,theC.botulinumtoxinC2II-channeldidnot share this feature of PA63. Cell-based experiments demonstrated that addition of an N-terminal His6-tag promoted also intoxicationofendothelialcellsbyC2IorEDINviaPA63.OurresultsrevealedthatadditionofHis6-tagstoseveralfactors increasetheirbindingpropertiestoPA63andenhancethepropertytointoxicatecells. Citation: Beitzinger C, Stefani C, Kronhardt A, Rolando M, Flatau G, et al. (2012) Role of N-Terminal His6-Tags in Binding and Efficient Translocation of PolypeptidesintoCellsUsingAnthraxProtectiveAntigen(PA).PLoSONE7(10):e46964.doi:1