by sphaeroidesgenomic DNA by PCR, using the primers5-AAGGTACCCTGCAGGCCCACGCCCTGAA-3and5-AAGATATCCACTGTGTCGTCTCCCAACT-3. 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 inE. 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/Significance == 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 preferred membrane protein. == Introduction == Membrane proteins (MPs) perform a wide range of essential biological functions and represent the largest class of protein drug targets (for reviews, observe[1][3]). Approximately 25% of all genes in both prokaryotes and eukaryotes code for MPs[4]and in humans 15% of these are G protein-coupled receptors (GPCRs)[2]. However, the vast majority of MPs still have no assigned function and only a little over 300 unique high-resolution 3D structures have Ethynylcytidine 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](http://blanco.biomol.uci.edu/mpstruc). This does not reflect the efforts deployed for the study of MPs in laboratories worldwide, but is an indication of the technical difficulties posed by the hydrophobic nature, generally low natural large quantity and intrinsic instability of these proteins. Obtaining sufficient amounts of MPs for functional and structural studies is the first Ethynylcytidine major bottleneck in their study[6][12]; and when expressed in heterologous systems, the proteins are frequentlyi) toxic for the host,ii) expressed at a very low level in a spatially-delimited membranous environment andiii) mis- or unfolded (and thus inactive)[13]. Protein overexpression entails 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 impossible to predict whether a protein of interest will express well, be easy to purify, be active or crystallise in any given experimental setup[14]. Consequently, it is often necessary to test various constructions in diverse expression hosts. Traditional cloning methods with Actual (Restriction Enzyme and Ligase) actions to generate multiple NIK expression plasmids (and constructs) 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 quick cloning of hundreds of genes and constructs simultaneously[14][17]. Among these, the Gateway technology[18], Creator[19]and the fragment exchange (FX) cloning[20]present the advantage of enabling subcloning of an open reading frame (ORF) 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[21], and several libraries from various ORFeome projects have been constructed using Gateway vectors[22][27]. Gateway Ethynylcytidine technology uses bacteriophage lambda Int/Xis/IHF recombination atattsites to transfer ORFs into vectors[28]. This divides the cloning process into three actions, as illustrated inFigure S1. In addition, Ethynylcytidine most of the expression vectors available can be made Gateway-compatible by inserting an adapter cassette containing Gateway-specific 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.
