Lactococcus lactis, an alternative system for functional expression of peripheral and intrinsic Arabidopsis membrane proteins.

<h4>Background</h4>Despite their functional and biotechnological importance, the study of membrane proteins remains difficult due to their hydrophobicity and their low natural abundance in cells. Furthermore, into established heterologous systems, these proteins are frequently only produ...

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Autores principales:	Annie Frelet-Barrand, Sylvain Boutigny, Lucas Moyet, Aurélien Deniaud, Daphné Seigneurin-Berny, Daniel Salvi, Florent Bernaudat, Pierre Richaud, Eva Pebay-Peyroula, Jacques Joyard, Norbert Rolland
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2010
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/90e4449ce2cc4361b349834bbf56a2f0
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spelling	oai:doaj.org-article:90e4449ce2cc4361b349834bbf56a2f02021-11-25T06:26:35ZLactococcus lactis, an alternative system for functional expression of peripheral and intrinsic Arabidopsis membrane proteins.1932-620310.1371/journal.pone.0008746https://doaj.org/article/90e4449ce2cc4361b349834bbf56a2f02010-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/20098692/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>Despite their functional and biotechnological importance, the study of membrane proteins remains difficult due to their hydrophobicity and their low natural abundance in cells. Furthermore, into established heterologous systems, these proteins are frequently only produced at very low levels, toxic and mis- or unfolded. Lactococcus lactis, a gram-positive lactic bacterium, has been traditionally used in food fermentations. This expression system is also widely used in biotechnology for large-scale production of heterologous proteins. Various expression vectors, based either on constitutive or inducible promoters, are available for this system. While previously used to produce bacterial and eukaryotic membrane proteins, the ability of this system to produce plant membrane proteins was until now not tested.<h4>Methodology/principal findings</h4>The aim of this work was to test the expression, in Lactococcus lactis, of either peripheral or intrinsic Arabidopsis membrane proteins that could not be produced, or in too low amount, using more classical heterologous expression systems. In an effort to easily transfer genes from Gateway-based Arabidopsis cDNA libraries to the L. lactis expression vector pNZ8148, we first established a cloning strategy compatible with Gateway entry vectors. Interestingly, the six tested Arabidopsis membrane proteins could be produced, in Lactococcus lactis, at levels compatible with further biochemical analyses. We then successfully developed solubilization and purification processes for three of these proteins. Finally, we questioned the functionality of a peripheral and an intrinsic membrane protein, and demonstrated that both proteins were active when produced in this system.<h4>Conclusions/significance</h4>Altogether, these data suggest that Lactococcus lactis might be an attractive system for the efficient and functional production of difficult plant membrane proteins.Annie Frelet-BarrandSylvain BoutignyLucas MoyetAurélien DeniaudDaphné Seigneurin-BernyDaniel SalviFlorent BernaudatPierre RichaudEva Pebay-PeyroulaJacques JoyardNorbert RollandPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 5, Iss 1, p e8746 (2010)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Annie Frelet-Barrand Sylvain Boutigny Lucas Moyet Aurélien Deniaud Daphné Seigneurin-Berny Daniel Salvi Florent Bernaudat Pierre Richaud Eva Pebay-Peyroula Jacques Joyard Norbert Rolland Lactococcus lactis, an alternative system for functional expression of peripheral and intrinsic Arabidopsis membrane proteins.
description	<h4>Background</h4>Despite their functional and biotechnological importance, the study of membrane proteins remains difficult due to their hydrophobicity and their low natural abundance in cells. Furthermore, into established heterologous systems, these proteins are frequently only produced at very low levels, toxic and mis- or unfolded. Lactococcus lactis, a gram-positive lactic bacterium, has been traditionally used in food fermentations. This expression system is also widely used in biotechnology for large-scale production of heterologous proteins. Various expression vectors, based either on constitutive or inducible promoters, are available for this system. While previously used to produce bacterial and eukaryotic membrane proteins, the ability of this system to produce plant membrane proteins was until now not tested.<h4>Methodology/principal findings</h4>The aim of this work was to test the expression, in Lactococcus lactis, of either peripheral or intrinsic Arabidopsis membrane proteins that could not be produced, or in too low amount, using more classical heterologous expression systems. In an effort to easily transfer genes from Gateway-based Arabidopsis cDNA libraries to the L. lactis expression vector pNZ8148, we first established a cloning strategy compatible with Gateway entry vectors. Interestingly, the six tested Arabidopsis membrane proteins could be produced, in Lactococcus lactis, at levels compatible with further biochemical analyses. We then successfully developed solubilization and purification processes for three of these proteins. Finally, we questioned the functionality of a peripheral and an intrinsic membrane protein, and demonstrated that both proteins were active when produced in this system.<h4>Conclusions/significance</h4>Altogether, these data suggest that Lactococcus lactis might be an attractive system for the efficient and functional production of difficult plant membrane proteins.
format	article
author	Annie Frelet-Barrand Sylvain Boutigny Lucas Moyet Aurélien Deniaud Daphné Seigneurin-Berny Daniel Salvi Florent Bernaudat Pierre Richaud Eva Pebay-Peyroula Jacques Joyard Norbert Rolland
author_facet	Annie Frelet-Barrand Sylvain Boutigny Lucas Moyet Aurélien Deniaud Daphné Seigneurin-Berny Daniel Salvi Florent Bernaudat Pierre Richaud Eva Pebay-Peyroula Jacques Joyard Norbert Rolland
author_sort	Annie Frelet-Barrand
title	Lactococcus lactis, an alternative system for functional expression of peripheral and intrinsic Arabidopsis membrane proteins.
title_short	Lactococcus lactis, an alternative system for functional expression of peripheral and intrinsic Arabidopsis membrane proteins.
title_full	Lactococcus lactis, an alternative system for functional expression of peripheral and intrinsic Arabidopsis membrane proteins.
title_fullStr	Lactococcus lactis, an alternative system for functional expression of peripheral and intrinsic Arabidopsis membrane proteins.
title_full_unstemmed	Lactococcus lactis, an alternative system for functional expression of peripheral and intrinsic Arabidopsis membrane proteins.
title_sort	lactococcus lactis, an alternative system for functional expression of peripheral and intrinsic arabidopsis membrane proteins.
publisher	Public Library of Science (PLoS)
publishDate	2010
url	https://doaj.org/article/90e4449ce2cc4361b349834bbf56a2f0
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Lactococcus lactis, an alternative system for functional expression of peripheral and intrinsic Arabidopsis membrane proteins.

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