Application of Ni(II)-Assisted Peptide Bond Hydrolysis to Non-Enzymatic Affinity Tag Removal 英文参考文献.docVIP

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Application of Ni(II)-Assisted Peptide Bond Hydrolysis to Non-Enzymatic Affinity Tag Removal 英文参考文献.doc

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Application of Ni(II)-Assisted Peptide Bond Hydrolysis to Non-Enzymatic Affinity Tag Removal 英文参考文献

ApplicationofNi(II)-AssistedPeptideBondHydrolysisto Non-EnzymaticAffinityTagRemoval EdytaKopera,AgnieszkaBelczyk-Ciesielska,WojciechBal* InstituteofBiochemistryandBiophysics,PolishAcademyofSciences,Warsaw,Poland Abstract Inthisstudy,wedemonstrateanon-enzymaticmethodforhydrolyticpeptidebondcleavage,appliedtotheremovalofan affinitytagfromarecombinantfusionprotein,SPI2-SRHWAP-His6.ThismethodisbasedonahighlyspecificNi(II)reaction with(S/T)XHZpeptidesequences.Itcanbeappliedfortheproteinattachedtoanaffinitycolumnortotheunboundprotein insolution.WestudiedtheeffectofpH,temperatureandNi(II)concentrationontheefficacyofcleavageanddevelopedan analytical protocol, which provides active protein with a 90% yield and ,100% purity. The method works well in the presenceofnon-ionicdetergents,DTTandGuHCl,thereforeprovidingaviablealternativeforcurrentlyusedtechniques. Citation: Kopera E, Belczyk-Ciesielska A, Bal W (2012) Application of Ni(II)-Assisted Peptide Bond Hydrolysis to Non-Enzymatic Affinity Tag Removal. PLoS ONE7(5):e36350.doi:10.1371/journal.pone.0036350 Editor:HendrikW.vanVeen,UniversityofCambridge,UnitedKingdom ReceivedFebruary2,2012;AcceptedApril2,2012;PublishedMay4,2012 Copyright: ? 2012 Kopera et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding:ThisstudywasfinancedbyPolishNationalCenterforResearchandDevelopment,grantno.KB/138/13749/IT1-B/U/08.Thefundershadnoroleinstudy design,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript. CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist. *E-mail:wbal@ibb.waw.pl Introduction unspecific reactions should be expected for this method, due to theabilityofCu(II)complexestogeneratereactiveoxygenspecies. 2+ Current techniques of protein science often require significant amounts of pure recombinant proteins. The aff