Abstract
BackgroundN-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 FindingsHere 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/SignificanceThe 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 quantitatively processed target protein that are sufficient for comprehensive biophysical and structural studies.
Highlights
N-myristoylation is the covalent attachment of myristic acid to an NH2-terminal glycine residue of a protein substrate via an amide bond [1]
Myristoylated Gag precursor polyprotein of the spleen necrosis virus is anchored in the plasma membrane during proteolytic cleavage
Nmyristoylation can have a strong impact on protein structure [6], e.g. it confers structural and thermal stability to the catalytic subunit of cAMP-dependent protein kinase [7,8]
Summary
N-myristoylation is the covalent attachment of myristic acid to an NH2-terminal glycine residue of a protein substrate via an amide bond [1]. Both the NMT and the target protein must be purified prior to the in vitro myristoylation and an additional purification step is necessary to remove the enzyme, unmodified protein and the fatty acid substrate after the reaction.
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