Heterologous expression of membrane proteins: choosing the appropriate host.
<h4>Background</h4>Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can...
Guardado en:
| Autores principales: | , , , , , , , , , , , , , , , , , |
|---|---|
| Formato: | article |
| Lenguaje: | EN |
| Publicado: |
Public Library of Science (PLoS)
2011
|
| Materias: | |
| Acceso en línea: | https://doaj.org/article/24e6ffec5e774736824ebc599b5bb6ee |
| Etiquetas: |
Agregar Etiqueta
Sin Etiquetas, Sea el primero en etiquetar este registro!
|
| id |
oai:doaj.org-article:24e6ffec5e774736824ebc599b5bb6ee |
|---|---|
| record_format |
dspace |
| spelling |
oai:doaj.org-article:24e6ffec5e774736824ebc599b5bb6ee2021-11-18T07:31:42ZHeterologous expression of membrane proteins: choosing the appropriate host.1932-620310.1371/journal.pone.0029191https://doaj.org/article/24e6ffec5e774736824ebc599b5bb6ee2011-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22216205/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein.<h4>Methodology/principal findings</h4>The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts.<h4>Conclusions/significance</h4>Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein.Florent BernaudatAnnie Frelet-BarrandNathalie PochonSébastien DementinPatrick HivinSylvain BoutignyJean-Baptiste RiouxDaniel SalviDaphné Seigneurin-BernyPierre RichaudJacques JoyardDavid PignolMonique SabatyThierry DesnosEva Pebay-PeyroulaElisabeth DarrouzetThierry VernetNorbert RollandPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 6, Iss 12, p e29191 (2011) |
| institution |
DOAJ |
| collection |
DOAJ |
| language |
EN |
| topic |
Medicine R Science Q |
| spellingShingle |
Medicine R Science Q Florent Bernaudat Annie Frelet-Barrand Nathalie Pochon Sébastien Dementin Patrick Hivin Sylvain Boutigny Jean-Baptiste Rioux Daniel Salvi Daphné Seigneurin-Berny Pierre Richaud Jacques Joyard David Pignol Monique Sabaty Thierry Desnos Eva Pebay-Peyroula Elisabeth Darrouzet Thierry Vernet Norbert Rolland Heterologous expression of membrane proteins: choosing the appropriate host. |
| description |
<h4>Background</h4>Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein.<h4>Methodology/principal findings</h4>The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts.<h4>Conclusions/significance</h4>Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein. |
| format |
article |
| author |
Florent Bernaudat Annie Frelet-Barrand Nathalie Pochon Sébastien Dementin Patrick Hivin Sylvain Boutigny Jean-Baptiste Rioux Daniel Salvi Daphné Seigneurin-Berny Pierre Richaud Jacques Joyard David Pignol Monique Sabaty Thierry Desnos Eva Pebay-Peyroula Elisabeth Darrouzet Thierry Vernet Norbert Rolland |
| author_facet |
Florent Bernaudat Annie Frelet-Barrand Nathalie Pochon Sébastien Dementin Patrick Hivin Sylvain Boutigny Jean-Baptiste Rioux Daniel Salvi Daphné Seigneurin-Berny Pierre Richaud Jacques Joyard David Pignol Monique Sabaty Thierry Desnos Eva Pebay-Peyroula Elisabeth Darrouzet Thierry Vernet Norbert Rolland |
| author_sort |
Florent Bernaudat |
| title |
Heterologous expression of membrane proteins: choosing the appropriate host. |
| title_short |
Heterologous expression of membrane proteins: choosing the appropriate host. |
| title_full |
Heterologous expression of membrane proteins: choosing the appropriate host. |
| title_fullStr |
Heterologous expression of membrane proteins: choosing the appropriate host. |
| title_full_unstemmed |
Heterologous expression of membrane proteins: choosing the appropriate host. |
| title_sort |
heterologous expression of membrane proteins: choosing the appropriate host. |
| publisher |
Public Library of Science (PLoS) |
| publishDate |
2011 |
| url |
https://doaj.org/article/24e6ffec5e774736824ebc599b5bb6ee |
| work_keys_str_mv |
AT florentbernaudat heterologousexpressionofmembraneproteinschoosingtheappropriatehost AT anniefreletbarrand heterologousexpressionofmembraneproteinschoosingtheappropriatehost AT nathaliepochon heterologousexpressionofmembraneproteinschoosingtheappropriatehost AT sebastiendementin heterologousexpressionofmembraneproteinschoosingtheappropriatehost AT patrickhivin heterologousexpressionofmembraneproteinschoosingtheappropriatehost AT sylvainboutigny heterologousexpressionofmembraneproteinschoosingtheappropriatehost AT jeanbaptisterioux heterologousexpressionofmembraneproteinschoosingtheappropriatehost AT danielsalvi heterologousexpressionofmembraneproteinschoosingtheappropriatehost AT daphneseigneurinberny heterologousexpressionofmembraneproteinschoosingtheappropriatehost AT pierrerichaud heterologousexpressionofmembraneproteinschoosingtheappropriatehost AT jacquesjoyard heterologousexpressionofmembraneproteinschoosingtheappropriatehost AT davidpignol heterologousexpressionofmembraneproteinschoosingtheappropriatehost AT moniquesabaty heterologousexpressionofmembraneproteinschoosingtheappropriatehost AT thierrydesnos heterologousexpressionofmembraneproteinschoosingtheappropriatehost AT evapebaypeyroula heterologousexpressionofmembraneproteinschoosingtheappropriatehost AT elisabethdarrouzet heterologousexpressionofmembraneproteinschoosingtheappropriatehost AT thierryvernet heterologousexpressionofmembraneproteinschoosingtheappropriatehost AT norbertrolland heterologousexpressionofmembraneproteinschoosingtheappropriatehost |
| _version_ |
1718423273688006656 |