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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...
Autores principales: | , , , |
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Formato: | Texto |
Lenguaje: | English |
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Public Library of Science
2010
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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. |
format | Text |
id | pubmed-2852408 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2010 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
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
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title_full | Single Vector System for Efficient N-myristoylation of Recombinant Proteins in E. coli
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title_fullStr | Single Vector System for Efficient N-myristoylation of Recombinant Proteins in E. coli
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title_full_unstemmed | Single Vector System for Efficient N-myristoylation of Recombinant Proteins in E. coli
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title_short | Single Vector System for Efficient N-myristoylation of Recombinant Proteins in E. coli
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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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