Semliki Forest virus vectors for overexpression of 101 G protein-coupled receptors in mammalian host cells

Semliki Forest virus vectors were applied for the evaluation of 101 G protein-coupled receptors in three mammalian cell lines. Western blotting demonstrated that 95 of the 101 tested GPCRs showed positive signals. A large number of the GPCRs were expressed at high levels suggesting receptor yields in the range of 1 mg/L or higher, suitable for structural biology applications. Specific binding assays on a selected number of GPCRs were carried out to compare the correlation between total and functional protein expression. Ligands and additives supplemented to the cell culture medium were evaluated for expression enhancement. Selected GPCRs were also expressed from mutant SFV vectors providing enhanced protein expression and reduced host cell toxicity in attempts to further improve receptor yields.     

Structural genomics on membrane proteins: comparison of more than 100 GPCRs in 3 expression systems

Production of recombinant receptors has been one of the major bottlenecks in structural biology on G protein-coupled receptors (GPCRs). The MePNet (Membrane Protein Network) was established to overexpress a large number of GPCRs in three major expression systems, based on Escherichia coli, Pichia pastoris and Semliki Forest virus (SFV) vectors. Evaluation by immunodetection demonstrated that 50% of a total of 103 GPCRs were expressed in bacterial inclusion bodies, 94% in yeast cell membranes and 95% in SFV-infected mammalian cells. The expression levels varied from low to high and the various GPCR families and subtypes were analyzed for their expressability in each expression system. More than 60% of the GPCRs were expressed at milligram levels or higher in one or several systems, compatible to structural biology applications. Functional activity was determined by binding assays in yeast and mammalian cells and the correlation between immunodetection and binding activity was analyzed.     

Large scale expression and purification of the mouse 5-HT3 receptor

Receptors of the Cys-loop family are central to neurotransmission and primary therapeutic targets. In order to decipher their gating and modulation mechanisms, structural data is essential. However, structural studies require large amounts of pure, functional receptors. Here, we present the expression and purification of the mouse serotonin 5-HT3 receptor to high purity and homogeneity levels. Inducible expression in human embryonic kidney 293 cells in suspension cultures with orbital shaking resulted in yields of 6–8 mg receptor per liter of culture. Affinity purification using a strep tag provided pure protein in active form. Further deglycosylation and removal of the purification tag led to a pentameric receptor after size-exclusion chromatography, at the milligram scale. This material is suitable for crystallography, as demonstrated by X-ray diffraction of receptor crystals at low resolution.     

Thermal Unfolding of a Mammalian Pentameric Ligand-gated Ion Channel Proceeds at Consecutive,Distinct Steps

Pentameric ligand-gated ion channels (LGICs) play an important role in fast synaptic signal transduction. Binding of agonists to the β-sheet-structured extracellular domain opens an ion channel in the transmembrane α-helical region of the LGIC. How the structurally distinct and distant domains are functionally coupled for such central transmembrane signaling processes remains an open question. To obtain detailed information about the stability of and the coupling between these different functional domains, we analyzed the thermal unfolding of a homopentameric LGIC, the 5-hydroxytryptamine receptor (ligand binding, secondary structure, accessibility of Trp and Cys residues, and aggregation), in plasma membranes as well as during detergent extraction, purification, and reconstitution into artificial lipid bilayers. We found a large loss in thermostability correlating with the loss of the lipid bilayer during membrane solubilization and purification. Thermal unfolding of the 5-hydroxytryptamine receptor occurred in consecutive steps at distinct protein locations. A loss of ligand binding was detected first, followed by formation of different transient low oligomeric states of receptor pentamers, followed by partial unfolding of helical parts of the protein, which finally lead to the formation receptor aggregates. Structural destabilization of the receptor in detergents could be partially reversed by reconstituting the receptor into lipid bilayers. Our results are important because they quantify the stability of LGICs during detergent extraction and purification and can be used to create stabilized receptor proteins for structural and functional studies.     

Comparisons between Geographically Diverse Samples of Carried Staphylococcus aureus

Approximately one-third of the human population is asymptomatically colonized by Staphylococcus aureus. However, much of the global diversity within the carriage populations remains uncharacterized, and it is unclear to what degree the variation is geographically partitioned. We isolated 300 carriage isolates from 1,531 adults contemporaneously in four countries: France, Algeria, Moldova, and Cambodia. All strains were characterized by multilocus sequence typing. Six clonal complexes (CCs) were present in all four samples (CC30, -45, -121, -15, -5, and -8). Analyses based on the genotype frequencies revealed the French and Algerian samples to be most similar and the Cambodian sample to be most distinct. While this pattern is consistent with likely rates of human migration and geographic distance, stochastic clonal expansion also contributes to regional differences. Phylogenetic analysis revealed a highly divergent and uncharacterized genotype (ST1223) within Cambodia. This lineage is related to CC75, which has previously been observed only in remote aboriginal populations in northern Australia.Although better known as an important human pathogen, Staphylococcus aureus is typically a commensal species and asymptomatically colonizes approximately one-third of the human population globally (18, 20, 29). This high carriage rate potentially represents a vast reservoir of as-yet-uncharacterized S. aureus diversity, an appreciation of which should shed light on the forces underpinning the diversification and dissemination of S. aureus. There are comparatively few studies examining spatial or temporal genotype distributions within carriage populations, and the extent of biogeographical structure is currently unclear, as is the level of discrimination which might be required to detect such structure.Multilocus sequence typing (MLST) has proved to be very successful as an epidemiological tool in that it delimits S. aureus in to a small number of widespread and discrete clonal complexes (CCs) (6, 8). These can be readily identified as clusters of related genotypes which have diversified radially from “founder” genotypes (9), and because this organism is largely clonal (8), assignments of isolates to these groups is broadly robust to the many different typing methods employed (4, 10, 27). The high level of divergence between these lineages suggests that they are relatively ancient and temporally stable (7), and it is possible that isolated host populations may have been colonized by different S. aureus lineages in the past. However, any footprints of geographical partitioning are likely to have been compromised by high rates of migration in recent times, due largely to the advent of air travel.Previous studies addressing the characterization of carried populations have tended to focus on samples from Western Europe or North America, and these have generally not provided strong evidence for geographical structuring. In a recent study using amplified fragment length polymorphism to compare the carried populations in Holland and North America, the authors noted considerable overlap between the samples, suggesting that they effectively constituted a single unstructured population (17). Similarly, independent MLST studies have revealed regional consistencies in Europe, such as the predominance of CC30 in the United Kingdom (8), Ireland (3), and Switzerland (25). Given the high rates of admixture within Europe and North America, the absence of obvious geographical structuring in the carried S. aureus population in these regions is perhaps not very surprising.Although they are currently scarce, current data from carriage populations outside of Europe or North America point to greater geographical structuring. For example, a sample of carried S. aureus recovered from Bamako, Mali, has recently been characterized, constituting the first such study of an African population (23). Although many of the previously characterized CCs were also present in this sample, the authors noted a high frequency (∼25%) of a single genotype, ST152, which is phylogenetically divergent and noted very rarely in Europe. The high frequency of ST152 in this population raises the possibility that this genotype is endemic to the Malian population and possibly elsewhere in sub-Saharan Africa. This in turn hints at greater geographical partitioning on a global scale, although more representative samples are clearly required. To address this, we generated MLST data from contemporaneous carriage samples recovered from four countries representing three continents: France (Western Europe), Moldova (Eastern Europe), Algeria (North Africa), and Cambodia (Southeast Asia). To our knowledge, this is the first time such a study has been carried out on samples from Eastern Europe, North Africa, or Southeast Asia. These data were therefore generated to uncover diversity within the global carriage population but also to understand further the extent to which geographical distance and host migration can explain regional differences.