Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

International audienceBACKGROUND: Membrane 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 FINDINGS: The 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/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 favourite membrane protein

Genetic evidence for the existence of two quinone related inhibitor binding sites in NADH-CoQ reductase

AbstractUsing the NADH-CoQ reductase of Rhodobacter capsulatus as a model for the mitochondrial Complex I, we have for the first time isolated bacterial mutants resistant to piericidin-A, a classical inhibitor of the mitochondrial enzyme. Their sensitivity to other inhibitors directed towards the quinone binding domain of complex I gives direct genetic evidence for the existence of two inhibitor binding sites.© 1977 Elsevier Science B.V. All rights reserved

Revisiting the CooJ family, a potential chaperone for nickel delivery to [NiFe]-carbon monoxide dehydrogenase

International audienceNickel insertion into nickel-dependent carbon monoxide dehydrogenase (CODH) represents a key step in the enzyme activation. This is the last step of the biosynthesis of the active site, which contains an atypical heteronuclear NiFe$_4$S$_4$ cluster known as the C-cluster. The enzyme maturation is performed by three accessory proteins, namely CooC, CooT and CooJ. Among them, CooJ from Rhodospirillum rubrum is a histidine-rich protein containing two distinct and spatially separated Ni(II)-binding sites: a N-terminal high affinity site (HAS) and a histidine tail at the C-terminus. In 46 CooJ homologues, the HAS motif was found to be strictly conserved with a H(W/F)XXHXXXH sequence. Here, a proteome database search identified at least 150 CooJ homologues and revealed distinct motifs for HAS, featuring 2, 3 or 4 histidines. The purification and biophysical characterization of three representative members of this protein family showed that they are all homodimers able to bind Ni(II) ions via one or two independent binding sites. Initially thought to be present only in R. rubrum, this study strongly suggests that CooJ could play a significant role in CODH maturation or in nickel homeostasis

The sodium/iodide symporter: State of the art of its molecular characterization

International audienceThe sodium/iodide symporter (NIS or SLC5A5) is an intrinsic membrane protein implicated in iodide uptake into thyroid follicular cells. It plays a crucial role in iodine metabolism and thyroid regulation and its function is widely exploited in the diagnosis and treatment of benign and malignant thyroid diseases. A great effort is currently being made to develop a NIS-based gene therapy also allowing the radiotreatment of nonthyroidal tumors. NIS is also expressed in other tissues, such as salivary gland, stomach and mammary gland during lactation, where its physiological role remains unclear. The molecular identity of the thyroid iodide transporter was elucidated approximately fifteen years ago. It belongs to the superfamily of sodium/solute symporters, SSS (and to the human transporter family, SLC5), and is composed of 13 transmembrane helices and 643 amino acid residues in humans. Knowledge concerning NIS structure/function relationship has been obtained by taking advantage of the high resolution structure of one member of the SSS family, the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT), and from studies of gene mutations leading to congenital iodine transport defects (ITD). This review will summarize current knowledge regarding the molecular characterization of NIS

Recent advances in cytochrome bc1: Inter monomer electronic communication?

AbstractThe ubihydroquinone: cytochrome c oxidoreductase, or cytochrome bc1, is a central component of photosynthetic and respiratory energy transduction pathways in many organisms. It contributes to the generation of membrane potential and proton gradient used for cellular energy production (ATP). The three-dimensional structures of cytochrome bc1 indicate that its two monomers are intertwined to form a symmetrical homodimer. This unusual architecture raises the issue of whether the monomers operate independently, or function cooperatively during the catalytic cycle of the enzyme. In this review, recent progresses achieved in our understanding of the mechanism of function of dimeric cytochrome bc1 are presented. New genetic approaches producing heterodimeric enzymes, and emerging insights related to the inter monomer electron transfer between the heme b cofactors of cytochrome bc1 are described

Development of a CO2-biomethanation reactor for producing methane from green H2

International audience"Power-to-Methane" approaches allow the storage and transport of green methane, produced from renewable energy and any CO2 source. In nature, some microorganisms, namely methanogens, can grow on CO2 and H-2 and produce pure methane via an ancestral process, the methanogenesis, under mild conditions (temperature, pressure, aqueous solvents. . .). These microorganisms are able to perform efficiently the Sabatier reaction (4H(2) + CO2 -> CH4 + 2H(2)O), using H-2 and CO2 as sole energy and carbon sources. Here, we developed a biomethanation reactor to culitvate a pure culture of Methanococcus maripaludis, a mesophilic methanogen growing rapidly at ambient temperature. A modular scalable and frugal 2 L-bubble column bioreactor was constructed to operate efficiently and autonomously for several weeks under a wide range of conditions. High H-2 conversion and methane yield higher than 90% could be reached. This high-performance, modular and robust bioreactor shows its potential for integration in outdoor systems coupling the conversion of alternative sources of green H-2 to fossil-free methane