Abstract
BackgroundMembrane 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.Methodology/Principal FindingsThe 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/SignificanceMost 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.
Highlights
Membrane proteins (MPs) perform a wide range of essential biological functions and represent the largest class of protein drug targets
As overexpression of membrane proteins is a challenge in itself, we focused our attention on the production step, and on the yields obtained in the various expression hosts tested
It is important to keep in mind that optimisation of expression conditions can greatly improve the yields of protein produced and it should be thoroughly undertaken after selecting the host
Summary
Membrane proteins (MPs) perform a wide range of essential biological functions and represent the largest class of protein drug targets (for reviews, see [1,2,3]). The vast majority of MPs still have no assigned function and only a little over 300 unique high-resolution 3D structures have been obtained for transmembrane proteins so far. Most of these structures are for bacterial and archaeal proteins, with only very few from eukaryotic systems [1,2,5] Traditional cloning methods with REaL (Restriction Enzyme and Ligase) steps to generate multiple expression plasmids (and constructs) are both labour-intensive and time-consuming. Gateway technology uses bacteriophage lambda Int/Xis/IHF recombination at att sites to transfer ORFs into vectors [28] This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein
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