Single Vector System for Efficient N-myristoylation of Recombinant Proteins in E. coli 英文参考文献.docVIP

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Single Vector System for Efficient N-myristoylation of Recombinant Proteins in E. coli 英文参考文献.doc

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Single Vector System for Efficient N-myristoylation of Recombinant Proteins in E. coli 英文参考文献

SingleVectorSystemforEfficientN-myristoylationof RecombinantProteinsinE.coli JulianM.Glu¨ck1,2,SilkeHoffmann1,BerndW.Koenig1,2,DieterWillbold1,2 * 1Institute of Structural Biology and Biophysics, Research Centre Ju¨lich, Ju¨lich, Germany, 2Institut fu¨r Physikalische Biologie, Heinrich-Heine-Universita¨t Du¨sseldorf, Du¨sseldorf,Germany Abstract Background:N-myristoylationisacrucialcovalentmodificationofnumerouseukaryoticandviralproteinsthatiscatalyzed by N-myristoyltransferase (NMT). Prokaryotes are lacking endogeneous NMT activity. Recombinant production of N- myristoylatedproteinsinE.colicellscanbeachievedbycoexpressionofheterologousNMTwiththetargetprotein.Inthe past,dualplasmidsystemswereusedforthispurpose. Methodology/Principal Findings: Here we describe a single vector system for efficient coexpression of substrate and enzymesuitableforproductionofco-orposttranslationallymodifiedproteins.TheapproachwasvalidatedusingtheHIV-1 Nefproteinasanexample.AsimpleandefficientprotocolforproductionofhighlypureandcompletelyN-myristoylated Nefispresented.Theyieldisabout20mgmyristoylatedNefperlitergrowthmedium. Conclusions/Significance: The single vector strategy allows diverse modifications of target proteins recombinantly coexpressed in E. coli with heterologous enzymes. The method is generally applicable and provides large amounts of quantitativelyprocessedtargetproteinthataresufficientforcomprehensivebiophysicalandstructuralstudies. Citation: Glu¨ck JM, Hoffmann S, Koenig BW, Willbold D (2010) Single Vector System for Efficient N-myristoylation of Recombinant Proteins in E. coli . PLoS ONE5(4):e10081.doi:10.1371/journal.pone.0010081 Editor:WalterS.Leal,UniversityofCaliforniaDavis,UnitedStatesofAmerica ReceivedJanuary26,2010;AcceptedMarch17,2010;PublishedApril9,2010 Copyright: ? 2010 Glu¨ck et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse,distribution,andreproductioninanymedium,pr