MARINE-EXPRESS: taking advantage of high throughput cloning and expression strategies for the post-genomic analysis of marine organisms

Background: The production of stable and soluble proteins is one of the most important steps prior to structural and functional studies of biological importance. We investigated the parallel production in a medium throughput strategy of genes coding for proteins from various marine organisms, using protocols that involved recombinatorial cloning, protein expression screening and batch purification. This strategy was applied in order to respond to the need for post-genomic validation of the recent success of a large number of marine genomic projects. Indeed, the upcoming challenge is to go beyond the bioinformatic data, since the bias introduced through the genomes of the so called model organisms leads to numerous proteins of unknown function in the still unexplored world of the oceanic organisms. Results: We present here the results of expression tests for 192 targets using a 96-well plate format. Genes were PCR amplified and cloned in parallel into expression vectors pFO4 and pGEX-4T-1, in order to express proteins N-terminally fused to a six-histidine-tag and to a GST-tag, respectively. Small-scale expression and purification permitted isolation of 84 soluble proteins and 34 insoluble proteins, which could also be used in refolding assays. Selected examples of proteins expressed and purified to a larger scale are presented. Conclusions: The objective of this program was to get around the bottlenecks of soluble, active protein expression and crystallization for post-genomic validation of a number of proteins that come from various marine organisms. Multiplying the constructions, vectors and targets treated in parallel is important for the success of a medium throughput strategy and considerably increases the chances to get rapid access to pure and soluble protein samples, needed for the subsequent biochemical characterizations. Our set up of a medium throughput strategy applied to genes from marine organisms had a mean success rate of 44% soluble protein expression from marine bacteria, archaea as well as eukaryotic organisms. This success rate compares favorably with other protein screening projects, particularly for eukaryotic proteins. Several purified targets have already formed the base for experiments aimed at post-genomic validation

Étude structurale et fonctionnelle de glycoside hydrolases et d'une iodo-péroxydase de la flavobactérie marine Zobellia galactanivorans, impliquées dans l'interaction avec les algues

The surface of algae is colonized by marine heterotrophic bacteria which entertain different trophic relationships with their host. These relationships may be of various types such as symbiosis, commensalism, saprophytic or pathogenic. In all cases the algal cell wall, which is mainly constituted by polysaccharides, is the first barrier bacteria have to interact with. Therefore, bacteria have developed sophisticated machineries (involving various glycoside hydrolases) to use the cell wall components as carbon source, but they also have to cope with algal defence reactions, which are also located in the cell wall of algae. Zobellia galactanivorans is a marine flavobacteria whose genome has recently been sequenced. It represents a model for the interaction between algae and bacteria. Indeed, 3 % of genes are involved in polysaccharide degradation, many of them organised in operon-like structures. Moreover, many enzymes present in the genome appear to be involved in original metabolisms, such as iodine utilization. My interest focused on the molecular aspects of three different factors involved in interaction of Z. galactanivorans with algae. The genome of Z. galactanivorans revealed two new sequences of glycosides hydrolases family 82 which are particular, due to their truncated sequences. Based on multiple sequence alignments, I designed site directed mutagenesis experiments, performed with the Alteromonas fortis enzyme, the first structural representant of iota-carrageenases. The results show that E245 (proton donor) and D247 (nucleophile) are the catalytic residues and that residues Q222, H281 and E310 also play crucial roles in the enzymatic reaction. Using a crystallographic approach, I was able to highlight the importance of a chloride ion in the formation of a water network, close to the active site. I also initiated the first biochemical and structural characterization of two members of a new GH family, located in operon-like gene organisations apparently involved in the degradation of sulphated polysaccharides. A third part of my thesis concerns the crystallographic structure determination of a first prokaryote, vanadium dependent iodoperoxydase, identified in the Z. galactanivorans genome. The structural analysis of this enzyme that may be involved in a detoxification reaction following the oxidative burst generated by algae, allows us to suggest a new evolution pattern for this type of enzymes.La surface des algues est colonisée par des bactéries marines hétérotrophes qui entretiennent avec leurs hôtes différentes relations trophiques. Zobellia galactanivorans, une flavobactérie marine, est un modèle de l'interaction entre les bactéries et les algues. 3 % des gènes de cette bactérie sont impliquées dans la dégradation de polysaccharides. De plus on retrouve, dans son génome, des enzymes participant à des métabolismes originaux comme celui de l'iode. Je me suis intéressé à 3 aspects moléculaires de l'interaction entre Z. galactanivorans et les algues. Premièrement le génome de Z. galactanivorans possède deux nouvelles iota-carraghénases de la famille GH 82, plus courtes que celles déjà décrites. Dans le but d'identifier la machinerie catalytique de ces enzymes, je me suis basé sur l'alignement multiple de l'ensemble des séquences de la famille GH 82 pour réaliser des expériences de mutagenèse dirigées sur la iotases d'Alteromonas fortis et ainsi expliquer le mécanisme catalytique de cette famille de glycoside hydrolase. J'ai également effectué une première caractérisation biochimique et structurale de deux membres d'une nouvelle famille de glycoside hydrolases, identifié, dans le génome de Z. galactanivorans, au sein d'opérons putatifs très probablement impliqués dans la dégradation de polysaccharides sulfatés. Enfin, j'ai résolu la première structure cristallographique d'une iodoperoxydase vanadium-dépendante d'origine bactérienne. Cette enzyme pourrait être impliquée dans un mécanisme de détoxification de la bactérie, suite à un ''burst oxydant'' généré par l'algue. Cette première structure permet de revoir et de discuter l'évolution de ces protéines

Etude structurale et fonctionnelle de glycoside hydrolases et d'une iodo-péroxydase de la flavobactérie marine Zobellia galactanivorans, impliquées dans l'interaction avec les algues

La surface des algues est colonisée par des bactéries marines hétérotrophes qui entretiennent avec leurs hôtes différentes relations trophiques. Zobellia galactanivorans, une flavobactérie marine, est un modèle de l'interaction entre les bactéries et les algues. 3 % des gènes de cette bactérie sont impliquées dans la dégradation de polysaccharides. De plus on retrouve, dans son génome, des enzymes participant à des métabolismes originaux comme celui de l'iode. Je me suis intéressé à 3 aspects moléculaires de l'interaction entre Z. galactanivorans et les algues. Premièrement le génome de Z. galactanivorans possède deux nouvelles iota-carraghénases de la famille GH 82, plus courtes que celles déjà décrites. Dans le but d'identifier la machinerie catalytique de ces enzymes, je me suis basé sur l'alignement multiple de l'ensemble des séquences de la famille GH 82 pour réaliser des expériences de mutagenèse dirigées sur la iotases d'Alteromonas fortis et ainsi expliquer le mécanisme catalytique de cette famille de glycoside hydrolase. J'ai également effectué une première caractérisation biochimique et structurale de deux membres d'une nouvelle famille de glycoside hydrolases, identifié, dans le génome de Z. galactanivorans, au sein d'opérons putatifs très probablement impliqués dans la dégradation de polysaccharides sulfatés. Enfin, j'ai résolu la première structure cristallographique d'une iodoperoxydase vanadium-dépendante d'origine bactérienne. Cette enzyme pourrait être impliquée dans un mécanisme de détoxification de la bactérie, suite à un ''burst oxydant'' généré par l'algue. Cette première structure permet de revoir et de discuter l'évolution de ces protéines.PARIS-BIUSJ-Physique recherche (751052113) / SudocROSCOFF-Observ.Océanol. (292393008) / SudocSudocFranceF

Cell-free production and characterisation of human uncoupling protein 1–3

The uncoupling proteins (UCPs) leak protons across the inner mitochondrial membrane, thus uncoupling the proton gradient from ATP synthesis. The main known physiological role for this is heat generation by UCP1 in brown adipose tissue. However, UCPs are also believed to be important for protection against reactive oxygen species, fine-tuning of metabolism and have been suggested to be involved in disease states such as obesity, diabetes and cancer. Structural studies of UCPs have long been hampered by difficulties in sample preparation with neither expression in yeast nor refolding from inclusion bodies in E. coli yielding sufficient amounts of pure and stable protein. In this study, we have developed a protocol for cell-free expression of human UCP1, 2 and 3, resulting in 1 mg pure protein per 20 mL of expression media. Lauric acid, a natural UCP ligand, significantly improved protein thermal stability and was therefore added during purification. Secondary structure characterisation using circular dichroism spectroscopy revealed the proteins to consist of mostly α-helices, as expected. All three UCPs were able to bind GDP, a well-known physiological inhibitor, as shown by the Fluorescence Resonance Energy Transfer (FRET) technique, suggesting that the proteins are in a natively folded state

The Bacterial Vanadium Iodoperoxidase from the Marine Flavobacteriaceae Zobellia galactanivorans Reveals Novel Molecular and Evolutionary Features of Halide Specificity in this Enzyme Family

International audienceVanadium haloperoxidases (VHPO) are key enzymes that oxidize halides and are involved in the biosynthesis of organo-halogens. Until now, only chloroperoxidases (VCPO) and bromoperoxidases (VBPO) have been characterized structurally, mainly from eukaryotic species. Three putative VHPO genes were predicted in the genome of the flavobacterium Zobellia galactanivorans, a marine bacterium associated with macroalgae. In a phylogenetic analysis, these putative bacterial VHPO were closely related to other VHPO from diverse bacterial phyla but clustered independently from eukaryotic algal VBPO and fungal VCPO. Two of these bacterial VHPO, heterogeneously produced in Escherichia coli, were found to be strictly specific for iodide oxidation. The crystal structure of one of these vanadium-dependent iodoperoxidases, Zg-VIPO1, was solved by multiwavelength anomalous diffraction at 1.8 Å, revealing a monomeric structure mainly folded into α-helices. This three-dimensional structure is relatively similar to those of VCPO of the fungus Curvularia inaequalis and of Streptomyces sp. and is superimposable onto the dimeric structure of algal VBPO. Surprisingly, the vanadate binding site of Zg-VIPO1 is strictly conserved with the fungal VCPO active site. Using site-directed mutagenesis, we showed that specific amino acids and the associated hydrogen bonding network around the vanadate center are essential for the catalytic properties and also the iodide specificity of Zg-VIPO1. Altogether, phylogeny and structure-function data support the finding that iodoperoxidase activities evolved independently in bacterial and algal lineages, and this sheds light on the evolution of the VHPO enzyme family

Exploring Selective Inhibition of the First Bromodomain of the Human Bromodomain and Extra-terminal Domain (BET) Proteins

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