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1. Heterologous expression of membrane proteins: choosing the appropriate host.

Author

Florent Bernaudat, Annie Frelet-Barrand, Nathalie Pochon, Sébastien Dementin, Patrick Hivin, Sylvain Boutigny, Jean-Baptiste Rioux, Daniel Salvi, Daphné Seigneurin-Berny, Pierre Richaud, Jacques Joyard, David Pignol, Monique Sabaty, Thierry Desnos, Eva Pebay-Peyroula, Elisabeth Darrouzet, Thierry Vernet, and Norbert Rolland

Subjects
Medicine, Science
Abstract

BackgroundMembrane 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 findingsThe 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/significanceMost 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.

Published
2011
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2. SeedUSoon: A New Software Program to Improve Seed Stock Management and Plant Line Exchanges between Research Laboratories

Author

Laurent Nussaume, Hélène Javot, Céline Charavay, Nathalie Pochon, Stéphane Segard, Institut de Biosciences et de Biotechnologies de Grenoble (ex-IRTSV) (BIG), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Biologie cellulaire et moléculaire des plantes et des bactéries (BCMPB), Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université de la Méditerranée - Aix-Marseille 2, Biologie végétale et microbiologie environnementale - UMR7265 (BVME), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Signalisation de l'Adaptation des Végétaux à l'Environnement (SAVE), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Université de la Méditerranée - Aix-Marseille 2-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Plant Environmental Physiology and Stress Signaling (PEPSS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes (UGA), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)

Subjects
0106 biological sciences, 0301 basic medicine, Computer science, Interface (Java), Data management, Context (language use), plant, Certification, Plant Science, 01 natural sciences, 03 medical and health sciences, Methods, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, genetics, genealogy, database, TRACE (psycholinguistics), Stock management, [INFO.INFO-DB]Computer Science [cs]/Databases [cs.DB], business.industry, software, Inheritance (genetic algorithm), food and beverages, Data science, Biotechnology, Material transfer agreement, 030104 developmental biology, MTA, business, seed, 010606 plant biology & botany
Abstract

International audience; Plant research is supported by an ever-growing collection of mutant or transgenic lines. In the past, a typical basic research laboratory would focus on only a few plant lines that were carefully isolated from collections of lines containing random mutations. The subsequent technological breakthrough in high-throughput sequencing, combined with novel and highly efficient mutagenesis techniques (including site-directed mutagenesis), has led to a recent exponential growth in plant line collections used by individual researchers. Tracking the generation and genetic properties of these genetic resources is thus becoming increasingly challenging for researchers. Another difficulty for researchers is controlling the use of seeds protected by a Material Transfer Agreement, as often only the original recipient of the seeds is aware of the existence of such documents. This situation can thus lead to difficult legal situations. Simultaneously, various institutions and the general public now demand more information about the use of genetically modified organisms (GMOs). In response, researchers are seeking new database solutions to address the triple challenge of research competition, legal constraints, and institutional/public demands. To help plant biology laboratories organize, describe, store, trace, and distribute their seeds, we have developed the new program SeedUSoon, with simplicity in mind. This software contains data management functions that allow the separate tracking of distinct mutations, even in successive crossings or mutagenesis. SeedUSoon reflects the biotechnological diversity of mutations and transgenes contained in any specific line, and the history of their inheritance. It can facilitate GMO certification procedures by distinguishing mutations on the basis of the presence/absence of a transgene, and by recording the technology used for their generation. Its interface can be customized to match the context and rules of any laboratory. In addition, SeedUSoon includes functions to help the laboratory protect intellectual property, export data, and facilitate seed exchange between laboratories. The SeedUSoon program, which is customizable to match individual practices and preferences, provides a powerful toolkit to plant laboratories searching for innovative approaches in laboratory management.

Published
2017
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3. Altered expression of cytosolic/nuclear HSC70-1 molecular chaperone affects development and abiotic stress tolerance in Arabidopsis thaliana

Author

Marie-Anne Roncato, Serge Chiarenza, Nathalie Pochon, Nathalie Leonhardt, Mathilde Clément, Audrey Creff, Anne-Claire Cazalé, Elena Marin, Laurent D. Noël, Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Biologie cellulaire et moléculaire des plantes et des bactéries (BCMPB), Université de la Méditerranée - Aix-Marseille 2-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Université de la Méditerranée - Aix-Marseille 2, Unité mixte de recherche interactions plantes-microorganismes, Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université de la Méditerranée - Aix-Marseille 2, Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées

Subjects
0106 biological sciences, SGT1, Physiology, Mutant, Arabidopsis, Y-RAY, AUXIN, Plant Science, 01 natural sciences, DnaK, Cytosol, Gene Expression Regulation, Plant, UV-C, HSP70, 2. Zero hunger, Abiotic component, 0303 health sciences, biology, Salt Tolerance, [SDV.BV.BOT]Life Sciences [q-bio]/Vegetal Biology/Botanics, Cell biology, Co-chaperone, Glucosyltransferases, embryonic structures, ARSENIC, Signal Transduction, HEAT SHOCK, animal structures, HSC70, Ultraviolet Rays, macromolecular substances, CADMIUM, 03 medical and health sciences, Stress, Physiological, Botany, 030304 developmental biology, Cell Nucleus, Indoleacetic Acids, Arabidopsis Proteins, Abiotic stress, HSC70 Heat-Shock Proteins, ARABIDOPSIS THALIANA, Meristem, biology.organism_classification, MERSITEM, Hsp70, Gamma Rays, Chaperone (protein), biology.protein, CO-CHAPERONE, 010606 plant biology & botany
Abstract

International audience; Molecular chaperones of the heat shock cognate 70 kDa (HSC70) family are highly conserved in all living organisms and assist nascent protein folding in normal physiological conditions as well as in biotic and abiotic stress conditions. In the absence of specific inhibitors or viable knockout mutants, cytosolic/nuclear HSC70-1 overexpression (OE) and mutants in the HSC70 co-chaperone SGT1 (suppressor of G2/M allele of skp1) were used as genetic tools to identify HSC70/SGT1 functions in Arabidopsis development and abiotic stress responses. HSC70-1 OE caused a reduction in root and shoot meristem activities, thus explaining the dwarfism of those plants. In addition, HSC70-1 OE did not impair auxin-dependent phenotypes, suggesting that SGT1 functions previously identified in auxin signalling are HSC70 independent. While responses to abiotic stimuli such as UV-C exposure, phosphate starvation, or seedling de-etiolation were not perturbed by HSC70-1 OE, it specifically conferred γ-ray hypersensitivity and tolerance to salt, cadmium (Cd), and arsenic (As). Cd and As perception was not perturbed, but plants overexpressing HSC70-1 accumulated less Cd, thus providing a possible molecular explanation for their tolerance phenotype. In summary, genetic evidence is provided for HSC70-1 involvement in a limited set of physiological processes, illustrating the essential and yet specific functions of this chaperone in development and abiotic stress responses in Arabidopsis.

Published
2009
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4. Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host.

Author

Bernaudat, Florent, Frelet-Barrand, Annie, Nathalie Pochon, Dementin, Sébastien, Hivin, Patrick, Boutigny, Sylvain, Rioux, Jean-Baptiste, Salvi, Daniel, Seigneurin-Berny, Daphné, Richaud, Pierre, Joyard, Jacques, Pignol, David, Sabaty, Monique, Desnos, Thierry, Pebay-Peyroula, Eva, Darrouzet, Elisabeth, Vernet, Thierry, and Rolland, Norbert

Subjects
MEMBRANE proteins, ESCHERICHIA coli, FUNGUS-bacterium relationships, HEREDITY, PROTEINS, BIOLOGICAL membranes
Abstract

Background: 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. [ABSTRACT FROM AUTHOR]

Published
2011
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