Heterologous expression of membrane proteins: choosing the appropriate host.
BACKGROUND
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.


RESULTS
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.


CONCLUSIONS
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.
Institut de Biologie Structurale Jean-Pierre Ebel, CEA, Grenoble, France. florent.bernaudat@ibs.fr
Florent
F
Bernaudat
Annie
A
Frelet-Barrand
Nathalie
N
Pochon
Sébastien
S
Dementin
Patrick
P
Hivin
Sylvain
S
Boutigny
Jean-Baptiste
JB
Rioux
Daniel
D
Salvi
Daphné
D
Seigneurin-Berny
Pierre
P
Richaud
Jacques
J
Joyard
David
D
Pignol
Monique
M
Sabaty
Thierry
T
Desnos
Eva
E
Pebay-Peyroula
Elisabeth
E
Darrouzet
Thierry
T
Vernet
Norbert
N
Rolland
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D018411:Spodoptera
D001426:Bacterial Proteins; D017931:DNA Primers; D008565:Membrane Proteins; D010940:Plant Proteins