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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 endogeneous NMT activity. Recombinant production of N-myristoylated proteins in E. coli cells can be achieved by coexpr...

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Autores principales: Glück, Julian M., Hoffmann, Silke, Koenig, Bernd W., Willbold, Dieter
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2852408/
https://www.ncbi.nlm.nih.gov/pubmed/20404920
http://dx.doi.org/10.1371/journal.pone.0010081
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author Glück, Julian M.
Hoffmann, Silke
Koenig, Bernd W.
Willbold, Dieter
author_facet Glück, Julian M.
Hoffmann, Silke
Koenig, Bernd W.
Willbold, Dieter
author_sort Glück, Julian M.
collection PubMed
description 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 and provides large amounts of quantitatively processed target protein that are sufficient for comprehensive biophysical and structural studies.
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spelling pubmed-28524082010-04-19 Single Vector System for Efficient N-myristoylation of Recombinant Proteins in E. coli Glück, Julian M. Hoffmann, Silke Koenig, Bernd W. Willbold, Dieter PLoS One Research Article 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 and provides large amounts of quantitatively processed target protein that are sufficient for comprehensive biophysical and structural studies. Public Library of Science 2010-04-09 /pmc/articles/PMC2852408/ /pubmed/20404920 http://dx.doi.org/10.1371/journal.pone.0010081 Text en Glück et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Glück, Julian M.
Hoffmann, Silke
Koenig, Bernd W.
Willbold, Dieter
Single Vector System for Efficient N-myristoylation of Recombinant Proteins in E. coli
title Single Vector System for Efficient N-myristoylation of Recombinant Proteins in E. coli
title_full Single Vector System for Efficient N-myristoylation of Recombinant Proteins in E. coli
title_fullStr Single Vector System for Efficient N-myristoylation of Recombinant Proteins in E. coli
title_full_unstemmed Single Vector System for Efficient N-myristoylation of Recombinant Proteins in E. coli
title_short Single Vector System for Efficient N-myristoylation of Recombinant Proteins in E. coli
title_sort single vector system for efficient n-myristoylation of recombinant proteins in e. coli
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2852408/
https://www.ncbi.nlm.nih.gov/pubmed/20404920
http://dx.doi.org/10.1371/journal.pone.0010081
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