Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

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.

Heterologous Expression of Moss Light-harvesting Complex Stress-related 1 (LHCSR1), the Chlorophyll a-Xanthophyll Pigment-protein Complex Catalyzing Non-photochemical Quenching, in Nicotiana sp.

“…Heterologous expression systems for functional membrane proteins have been previously reported based on bacteria, cyanobacteria, and yeasts (43,44). However, the case of pigmentbinding proteins of the LHC family is more complex because of the need for multiple chromophores essential for the folding of the holoprotein.…”

Improved membrane protein expression in Lactococcus lactis by fusion to Mistic

“…The expression level of pkjDes4 was up to 4.4 % of total membrane proteins, which was high compared to that of other eukaryotic membrane proteins expressed in L. lactis (Kunji et al, 2003;Bernaudat et al, 2011). However, the percentage might be underestimated, as the membrane is readily contaminated with soluble and peripheral membrane proteins (up to 50 %) (Kunji et al, 2003).…”

“…To date, dozens of membrane proteins have been successfully overexpressed in L. lactis, including several eukaryotic membrane proteins (Kunji et al, 2003;Wieczorek & Martin, 2010;Steen et al, 2011;Marreddy et al, 2011;Bernaudat et al, 2011). However, the majority of prokaryotic membrane proteins were from L. lactis sources, and the eukaryotic membrane proteins were, to a large degree, limited to membrane transporters or carriers.…”

“…In this respect, Lactococcus lactis has been successfully used as an expression host because of the ideal properties it possesses: single-membrane structure, a strong and tightly regulated promoter system, mild proteolytic activity and non-inclusion body targeting (Kunji et al, 2003). Recently, a dozen membrane proteins, including transporting proteins, receptors and a quinone oxidoreductase, have been successfully overexpressed in lactococcal hosts (Kunji et al, 2003;Wieczorek & Martin, 2010;Steen et al, 2011;Marreddy et al, 2011;Bernaudat et al, 2011).…”

Overview of Recent Progress in Protein-Expression Technologies for Small-Molecule Screening

“…11,97,98 Even though approximately 25% of all prokaryotic and eukaryotic genes encode membrane proteins, most still are considered proteins of unknown function, and only slightly more than 300 such proteins have high-resolution crystal structures. 99 It is clear from these observations that the overexpression of integral membrane proteins to sufficient levels to enable downstream purification and use in screening presents a unique set of challenges. These proteins are highly hydrophobic and prefer to exist in a lipid bilayer environment.…”