single vector system for efficient n-myristoylation of recombinant proteins in e. coli单矢量系统高效n-myristoylation重组蛋白在大肠杆菌.pdfVIP

Single Vector System for Efficient N-myristoylation of Recombinant Proteins in E. coli ¨ 1,2 1 1,2 1,2 Julian M. Gluck , Silke Hoffmann , Bernd W. Koenig , Dieter Willbold * ¨ ¨ ¨ ¨ ¨ 1 Institute of Structural Biology and Biophysics, Research Centre Julich, Julich, Germany, 2 Institut fur Physikalische Biologie, Heinrich-Heine-Universitat Dusseldorf, ¨ Dusseldorf, Germany Abstract Background: N-myristoylation is a crucial covalent modification of numerous eukaryotic and viral proteins that is catalyzed by N-myristoyltransferase (NMT). Prokaryotes are lacking endogeneous NMT activity. Recombinant production of N- myristoylated proteins in E. coli cells can be achieved by coexpression of heterologous NMT with the target protein. In the past, dual plasmid systems were used for this purpose. Methodology/Principal Findings: Here we describe a single vector system for efficient coexpression of substrate and enzyme suitable for production of co- or posttranslationally modified proteins. The approach was validated using the HIV-1 Nef protein as an example. A simple and efficient protocol for production of highly pure and completely N-myristoylated Nef is presented. The yield is about 20 mg myristoylated Nef per liter growth medium. 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