Single vector system for efficient N-myristoylation of recombinant proteins in E. coli.
BACKGROUND
N-myristoylation is a crucial covalent modification of numerous eukaryotic and viral proteins that is catalyzed by N-myristoyltransferase (NMT). Prokaryotes are lacking endogenous 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.


RESULTS
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
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 quantitatively processed target protein that are sufficient for comprehensive biophysical and structural studies.
Glück
Julian M
JM
Institute of Structural Biology and Biophysics, Research Centre Jülich, Jülich, Germany.
Hoffmann
Silke
S
Koenig
Bernd W
BW
Willbold
Dieter
D
D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D008722:Methods; D009227:Myristic Acids / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism
D009227:Myristic Acids; D011994:Recombinant Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase
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