However, 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. This does not reflect the efforts deployed for the study of MPs in laboratories worldwide, but is an indication of the technical challenges posed by the hydrophobic nature, generally low natural abundance and intrinsic instability of these proteins. Obtaining sufficient amounts of MPs for functional and structural studies is the first major bottleneck in their study ; and when expressed in heterologous systems, the proteins are frequently i) toxic for the host, ii) expressed at a very low level in a spatiallydelimited membranous environment and iii) mis- or unfolded. Protein overexpression involves three elements: a gene, a vector and an expression host. The appropriate combination of these elements maximises the amount and quality of protein produced. However, since proteins are very diverse in structure and physico-chemical properties, it is R428 impossible to predict whether a protein of interest will express well, be easy to purify, be active or crystallise in any given experimental setup. Consequently, it is often necessary to test various constructions in diverse expression hosts. Traditional cloning methods with REaL steps to generate multiple expression plasmids are both labour-intensive and time-consuming. This makes them incompatible with a massively parallel strategy of expression screening. However, over the past few years, several recombinatorial cloning systems have been developed to allow rapid cloning of hundreds of genes and constructs simultaneously. Among these, the Gateway technology, Creator and the fragment exchange cloning present the advantage of enabling subcloning of an open reading frame into multiple expression vectors. Even if often adding extra-sequences to the proteins, Gateway is the most widely used and this technique has already been successfully exploited for high-throughput cloning of MPs, and several libraries from various ORFeome projects have been constructed using Gateway vectors. Gateway technology uses bacteriophage lambda Int/Xis/IHF recombination at att sites to transfer ORFs into vectors. This divides the cloning procedure into three steps, as illustrated in Figure S1. In addition, most of the expression vectors available can be made Gatewaycompatible by inserting an adapter cassette containing Gatewayspecific recombination sites. Once the expression vectors are obtained, production of the target proteins can be tested in different prokaryotic and eukaryotic expression systems suitable for overexpression of MPs. However, each of these systems has pros and cons, and the choice of the appropriate expression system often remains empirical, particularly with regard to the levels of functional protein expression. In the following paragraphs, we will briefly present the host systems tested in this study. The cloning strategy chosen for this project, based on Gateway technology, enabled us to obtain expression vectors for the different systems in a convenient and very efficient manner.