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1. Concentration vs. Optical Density of ESKAPEE Bacteria: A Method to Determine the Optimum Measurement Wavelength

Author: Bruno Wacogne, Marine Belinger Podevin, Naïs Vaccari, Claudia Koubevi, Céline Codjiová, Emilie Gutierrez, Lucie Davoine, Marjorie Robert-Nicoud, Alain Rouleau, and Annie Frelet-Barrand
Subjects: ESKAPEE bacteria, optical density, white light spectroscopy, absorbance spectra, spectra processing, Chemical technology, TP1-1185
Abstract: Optical density measurement has been used for decades to determine the microorganism concentration and more rarely for mammalian cells. Although this measurement can be carried out at any wavelength, studies report a limited number of measurement wavelengths, mainly around 600 nm, and no consensus seems to be emerging to propose an objective method for determining the optimum measurement wavelength for each microorganism. In this article, we propose a method for analyzing the absorbance spectra of ESKAPEE bacteria and determining the optimum measurement wavelength for each of them. The method is based on the analysis of the signal-to-noise ratio of the relationships between concentrations and optical densities when the measurement wavelength varies over the entire spectral range of the absorbance spectra measured for each bacterium. These optimum wavelengths range from 612 nm for Enterococcus faecium to 705 nm for Acinetobacter baumannii. The method can be directly applied to any bacteria, any culture method, and also to any biochemical substance with an absorbance spectrum without any particular feature such as an identified maximum.
Published: 2024
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2. Absorption/Attenuation Spectral Description of ESKAPEE Bacteria: Application to Seeder-Free Culture Monitoring, Mammalian T-Cell and Bacteria Mixture Analysis and Contamination Description

Author: Bruno Wacogne, Marine Belinger Podevin, Naïs Vaccari, Claudia Koubevi, Céline Codjiová, Emilie Gutierrez, Pauline Bourgeois, Lucie Davoine, Marjorie Robert-Nicoud, Alain Rouleau, and Annie Frelet-Barrand
Subjects: white light spectroscopy, absorption spectra, ESKAPEE bacteria, Lactococcus lactis, co-culture monitoring, mammalian T-cells, Chemical technology, TP1-1185
Abstract: Despite numerous innovations, measuring bacteria concentrations on a routine basis is still time consuming and ensuring accurate measurements requires careful handling. Furthermore, it often requires sampling small volumes of bacteria suspensions which might be poorly representative of the real bacteria concentration. In this paper, we propose a spectroscopy measurement method based on a description of the absorption/attenuation spectra of ESKAPEE bacteria. Concentrations were measured with accuracies less than 2%. In addition, mixing the mathematical description of the absorption/attenuation spectra of mammalian T-cells and bacteria allows for the simultaneous measurements of both species’ concentrations. This method allows real-time, sampling-free and seeder-free measurement and can be easily integrated into a closed-system environment.
Published: 2023
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3. Absorption Spectra Description for T-Cell Concentrations Determination and Simultaneous Measurements of Species during Co-Cultures

Author: Bruno Wacogne, Naïs Vaccari, Claudia Koubevi, Marine Belinger-Podevin, Marjorie Robert-Nicoud, Alain Rouleau, and Annie Frelet-Barrand
Subjects: T-cell culture, co-culture monitoring, white light spectroscopy, advanced therapy medicinal product, Chemical technology, TP1-1185
Abstract: Advanced Therapy Medicinal Products are promising drugs for patients in therapeutic impasses. Their complex fabrication process implies regular quality controls to monitor cell concentration. Among the different methods available, optical techniques offer several advantages. Our study aims to measure cell concentration in real time in a potential closed-loop environment using white light spectroscopy and to test the possibility of simultaneously measuring concentrations of several species. By analyzing the shapes of the absorption spectra, this system allowed the quantification of T-cells with an accuracy of about 3% during 30 h of cultivation monitoring and 26 h of doubling time, coherent with what is expected for normal cell culture. Moreover, our system permitted concentration measurements for two species in reconstructed co-cultures of T-cells and Candida albicans yeasts. This method can now be applied to any single or co-culture, it allows real-time monitoring, and can be easily integrated into a closed system.
Published: 2022
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4. Lactococcus lactis, an Attractive Cell Factory for the Expression of Functional Membrane Proteins

Author: Annie Frelet-Barrand
Subjects: Lactococcus lactis, membrane proteins, NICE system, Microbiology, QR1-502
Abstract: Membrane proteins play key roles in most crucial cellular processes, ranging from cell-to-cell communication to signaling processes. Despite recent improvements, the expression of functionally folded membrane proteins in sufficient amounts for functional and structural characterization remains a challenge. Indeed, it is still difficult to predict whether a protein can be overproduced in a functional state in some expression system(s), though studies of high-throughput screens have been published in recent years. Prokaryotic expression systems present several advantages over eukaryotic ones. Among them, Lactococcus lactis (L. lactis) has emerged in the last two decades as a good alternative expression system to E. coli. The purpose of this chapter is to describe L. lactis and its tightly inducible system, NICE, for the effective expression of membrane proteins from both prokaryotic and eukaryotic origins.
Published: 2022
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5. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

Author: Clotilde Théry, Kenneth W Witwer, Elena Aikawa, Maria Jose Alcaraz, Johnathon D Anderson, Ramaroson Andriantsitohaina, Anna Antoniou, Tanina Arab, Fabienne Archer, Georgia K Atkin-Smith, D Craig Ayre, Jean-Marie Bach, Daniel Bachurski, Hossein Baharvand, Leonora Balaj, Shawn Baldacchino, Natalie N Bauer, Amy A Baxter, Mary Bebawy, Carla Beckham, Apolonija Bedina Zavec, Abderrahim Benmoussa, Anna C Berardi, Paolo Bergese, Ewa Bielska, Cherie Blenkiron, Sylwia Bobis-Wozowicz, Eric Boilard, Wilfrid Boireau, Antonella Bongiovanni, Francesc E Borràs, Steffi Bosch, Chantal M Boulanger, Xandra Breakefield, Andrew M Breglio, Meadhbh Á Brennan, David R Brigstock, Alain Brisson, Marike LD Broekman, Jacqueline F Bromberg, Paulina Bryl-Górecka, Shilpa Buch, Amy H Buck, Dylan Burger, Sara Busatto, Dominik Buschmann, Benedetta Bussolati, Edit I Buzás, James Bryan Byrd, Giovanni Camussi, David RF Carter, Sarah Caruso, Lawrence W Chamley, Yu-Ting Chang, Chihchen Chen, Shuai Chen, Lesley Cheng, Andrew R Chin, Aled Clayton, Stefano P Clerici, Alex Cocks, Emanuele Cocucci, Robert J Coffey, Anabela Cordeiro-da-Silva, Yvonne Couch, Frank AW Coumans, Beth Coyle, Rossella Crescitelli, Miria Ferreira Criado, Crislyn D’Souza-Schorey, Saumya Das, Amrita Datta Chaudhuri, Paola de Candia, Eliezer F De Santana, Olivier De Wever, Hernando A del Portillo, Tanguy Demaret, Sarah Deville, Andrew Devitt, Bert Dhondt, Dolores Di Vizio, Lothar C Dieterich, Vincenza Dolo, Ana Paula Dominguez Rubio, Massimo Dominici, Mauricio R Dourado, Tom AP Driedonks, Filipe V Duarte, Heather M Duncan, Ramon M Eichenberger, Karin Ekström, Samir EL Andaloussi, Celine Elie-Caille, Uta Erdbrügger, Juan M Falcón-Pérez, Farah Fatima, Jason E Fish, Miguel Flores-Bellver, András Försönits, Annie Frelet-Barrand, Fabia Fricke, Gregor Fuhrmann, Susanne Gabrielsson, Ana Gámez-Valero, Chris Gardiner, Kathrin Gärtner, Raphael Gaudin, Yong Song Gho, Bernd Giebel, Caroline Gilbert, Mario Gimona, Ilaria Giusti, Deborah CI Goberdhan, André Görgens, Sharon M Gorski, David W Greening, Julia Christina Gross, Alice Gualerzi, Gopal N Gupta, Dakota Gustafson, Aase Handberg, Reka A Haraszti, Paul Harrison, Hargita Hegyesi, An Hendrix, Andrew F Hill, Fred H Hochberg, Karl F Hoffmann, Beth Holder, Harry Holthofer, Baharak Hosseinkhani, Guoku Hu, Yiyao Huang, Veronica Huber, Stuart Hunt, Ahmed Gamal-Eldin Ibrahim, Tsuneya Ikezu, Jameel M Inal, Mustafa Isin, Alena Ivanova, Hannah K Jackson, Soren Jacobsen, Steven M Jay, Muthuvel Jayachandran, Guido Jenster, Lanzhou Jiang, Suzanne M Johnson, Jennifer C Jones, Ambrose Jong, Tijana Jovanovic-Talisman, Stephanie Jung, Raghu Kalluri, Shin-ichi Kano, Sukhbir Kaur, Yumi Kawamura, Evan T Keller, Delaram Khamari, Elena Khomyakova, Anastasia Khvorova, Peter Kierulf, Kwang Pyo Kim, Thomas Kislinger, Mikael Klingeborn, David J Klinke, Miroslaw Kornek, Maja M Kosanović, Árpád Ferenc Kovács, Eva-Maria Krämer-Albers, Susanne Krasemann, Mirja Krause, Igor V Kurochkin, Gina D Kusuma, Sören Kuypers, Saara Laitinen, Scott M Langevin, Lucia R Languino, Joanne Lannigan, Cecilia Lässer, Louise C Laurent, Gregory Lavieu, Elisa Lázaro-Ibáñez, Soazig Le Lay, Myung-Shin Lee, Yi Xin Fiona Lee, Debora S Lemos, Metka Lenassi, Aleksandra Leszczynska, Isaac TS Li, Ke Liao, Sten F Libregts, Erzsebet Ligeti, Rebecca Lim, Sai Kiang Lim, Aija Linē, Karen Linnemannstöns, Alicia Llorente, Catherine A Lombard, Magdalena J Lorenowicz, Ákos M Lörincz, Jan Lötvall, Jason Lovett, Michelle C Lowry, Xavier Loyer, Quan Lu, Barbara Lukomska, Taral R Lunavat, Sybren LN Maas, Harmeet Malhi, Antonio Marcilla, Jacopo Mariani, Javier Mariscal, Elena S Martens-Uzunova, Lorena Martin-Jaular, M Carmen Martinez, Vilma Regina Martins, Mathilde Mathieu, Suresh Mathivanan, Marco Maugeri, Lynda K McGinnis, Mark J McVey, David G Meckes, Katie L Meehan, Inge Mertens, Valentina R Minciacchi, Andreas Möller, Malene Møller Jørgensen, Aizea Morales-Kastresana, Jess Morhayim, François Mullier, Maurizio Muraca, Luca Musante, Veronika Mussack, Dillon C Muth, Kathryn H Myburgh, Tanbir Najrana, Muhammad Nawaz, Irina Nazarenko, Peter Nejsum, Christian Neri, Tommaso Neri, Rienk Nieuwland, Leonardo Nimrichter, John P Nolan, Esther NM Nolte-’t Hoen, Nicole Noren Hooten, Lorraine O’Driscoll, Tina O’Grady, Ana O’Loghlen, Takahiro Ochiya, Martin Olivier, Alberto Ortiz, Luis A Ortiz, Xabier Osteikoetxea, Ole Østergaard, Matias Ostrowski, Jaesung Park, D. Michiel Pegtel, Hector Peinado, Francesca Perut, Michael W Pfaffl, Donald G Phinney, Bartijn CH Pieters, Ryan C Pink, David S Pisetsky, Elke Pogge von Strandmann, Iva Polakovicova, Ivan KH Poon, Bonita H Powell, Ilaria Prada, Lynn Pulliam, Peter Quesenberry, Annalisa Radeghieri, Robert L Raffai, Stefania Raimondo, Janusz Rak, Marcel I Ramirez, Graça Raposo, Morsi S Rayyan, Neta Regev-Rudzki, Franz L Ricklefs, Paul D Robbins, David D Roberts, Silvia C Rodrigues, Eva Rohde, Sophie Rome, Kasper MA Rouschop, Aurelia Rughetti, Ashley E Russell, Paula Saá, Susmita Sahoo, Edison Salas-Huenuleo, Catherine Sánchez, Julie A Saugstad, Meike J Saul, Raymond M Schiffelers, Raphael Schneider, Tine Hiorth Schøyen, Aaron Scott, Eriomina Shahaj, Shivani Sharma, Olga Shatnyeva, Faezeh Shekari, Ganesh Vilas Shelke, Ashok K Shetty, Kiyotaka Shiba, Pia R-M Siljander, Andreia M Silva, Agata Skowronek, Orman L Snyder, Rodrigo Pedro Soares, Barbara W Sódar, Carolina Soekmadji, Javier Sotillo, Philip D Stahl, Willem Stoorvogel, Shannon L Stott, Erwin F Strasser, Simon Swift, Hidetoshi Tahara, Muneesh Tewari, Kate Timms, Swasti Tiwari, Rochelle Tixeira, Mercedes Tkach, Wei Seong Toh, Richard Tomasini, Ana Claudia Torrecilhas, Juan Pablo Tosar, Vasilis Toxavidis, Lorena Urbanelli, Pieter Vader, Bas WM van Balkom, Susanne G van der Grein, Jan Van Deun, Martijn JC van Herwijnen, Kendall Van Keuren-Jensen, Guillaume van Niel, Martin E van Royen, Andre J van Wijnen, M Helena Vasconcelos, Ivan J Vechetti, Tiago D Veit, Laura J Vella, Émilie Velot, Frederik J Verweij, Beate Vestad, Jose L Viñas, Tamás Visnovitz, Krisztina V Vukman, Jessica Wahlgren, Dionysios C Watson, Marca HM Wauben, Alissa Weaver, Jason P Webber, Viktoria Weber, Ann M Wehman, Daniel J Weiss, Joshua A Welsh, Sebastian Wendt, Asa M Wheelock, Zoltán Wiener, Leonie Witte, Joy Wolfram, Angeliki Xagorari, Patricia Xander, Jing Xu, Xiaomei Yan, María Yáñez-Mó, Hang Yin, Yuana Yuana, Valentina Zappulli, Jana Zarubova, Vytautas Žėkas, Jian-ye Zhang, Zezhou Zhao, Lei Zheng, Alexander R Zheutlin, Antje M Zickler, Pascale Zimmermann, Angela M Zivkovic, Davide Zocco, and Ewa K Zuba-Surma
Subjects: extracellular vesicles, exosomes, ectosomes, microvesicles, minimal information requirements, guidelines, standardization, microparticles, rigor, reproducibility, Cytology, QH573-671
Abstract: The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.
Published: 2018
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6. Ectopic Neo-Formed Intracellular Membranes in Escherichia coli: A Response to Membrane Protein-Induced Stress Involving Membrane Curvature and Domains

Author: Nadège Jamin, Manuel Garrigos, Christine Jaxel, Annie Frelet-Barrand, and Stéphane Orlowski
Subjects: intracellular membranes, Escherichia coli, membrane domains, vesicles, tubules, membrane curvature, membrane protein overexpression, Microbiology, QR1-502
Abstract: Bacterial cytoplasmic membrane stress induced by the overexpression of membrane proteins at high levels can lead to formation of ectopic intracellular membranes. In this review, we report the various observations of such membranes in Escherichia coli, compare their morphological and biochemical characterizations, and we analyze the underlying molecular processes leading to their formation. Actually, these membranes display either vesicular or tubular structures, are separated or connected to the cytoplasmic membrane, present mono- or polydispersed sizes and shapes, and possess ordered or disordered arrangements. Moreover, their composition differs from that of the cytoplasmic membrane, with high amounts of the overexpressed membrane protein and altered lipid-to-protein ratio and cardiolipin content. These data reveal the importance of membrane domains, based on local specific lipid–protein and protein–protein interactions, with both being crucial for local membrane curvature generation, and they highlight the strong influence of protein structure. Indeed, whether the cylindrically or spherically curvature-active proteins are actively curvogenic or passively curvophilic, the underlying molecular scenarios are different and can be correlated with the morphological features of the neo-formed internal membranes. Delineating these molecular mechanisms is highly desirable for a better understanding of protein–lipid interactions within membrane domains, and for optimization of high-level membrane protein production in E. coli.
Published: 2018
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7. Oligomeric status and nucleotide binding properties of the plastid ATP/ADP transporter 1: toward a molecular understanding of the transport mechanism.

Author: Aurélien Deniaud, Pankaj Panwar, Annie Frelet-Barrand, Florent Bernaudat, Céline Juillan-Binard, Christine Ebel, Norbert Rolland, and Eva Pebay-Peyroula
Subjects: Medicine, Science
Abstract: BACKGROUND: Chloroplast ATP/ADP transporters are essential to energy homeostasis in plant cells. However, their molecular mechanism remains poorly understood, primarily due to the difficulty of producing and purifying functional recombinant forms of these transporters. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we describe an expression and purification protocol providing good yields and efficient solubilization of NTT1 protein from Arabidopsis thaliana. By biochemical and biophysical analyses, we identified the best detergent for solubilization and purification of functional proteins, LAPAO. Purified NTT1 was found to accumulate as two independent pools of well folded, stable monomers and dimers. ATP and ADP binding properties were determined, and Pi, a co-substrate of ADP, was confirmed to be essential for nucleotide steady-state transport. Nucleotide binding studies and analysis of NTT1 mutants lead us to suggest the existence of two distinct and probably inter-dependent binding sites. Finally, fusion and deletion experiments demonstrated that the C-terminus of NTT1 is not essential for multimerization, but probably plays a regulatory role, controlling the nucleotide exchange rate. CONCLUSIONS/SIGNIFICANCE: Taken together, these data provide a comprehensive molecular characterization of a chloroplast ATP/ADP transporter.
Published: 2012
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8. 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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9. Lactococcus lactis, an alternative system for functional expression of peripheral and intrinsic Arabidopsis membrane proteins.

Author: Annie Frelet-Barrand, Sylvain Boutigny, Lucas Moyet, Aurélien Deniaud, Daphné Seigneurin-Berny, Daniel Salvi, Florent Bernaudat, Pierre Richaud, Eva Pebay-Peyroula, Jacques Joyard, and Norbert Rolland
Subjects: Medicine, Science
Abstract: BackgroundDespite their functional and biotechnological importance, the study of membrane proteins remains difficult due to their hydrophobicity and their low natural abundance in cells. Furthermore, into established heterologous systems, these proteins are frequently only produced at very low levels, toxic and mis- or unfolded. Lactococcus lactis, a gram-positive lactic bacterium, has been traditionally used in food fermentations. This expression system is also widely used in biotechnology for large-scale production of heterologous proteins. Various expression vectors, based either on constitutive or inducible promoters, are available for this system. While previously used to produce bacterial and eukaryotic membrane proteins, the ability of this system to produce plant membrane proteins was until now not tested.Methodology/principal findingsThe aim of this work was to test the expression, in Lactococcus lactis, of either peripheral or intrinsic Arabidopsis membrane proteins that could not be produced, or in too low amount, using more classical heterologous expression systems. In an effort to easily transfer genes from Gateway-based Arabidopsis cDNA libraries to the L. lactis expression vector pNZ8148, we first established a cloning strategy compatible with Gateway entry vectors. Interestingly, the six tested Arabidopsis membrane proteins could be produced, in Lactococcus lactis, at levels compatible with further biochemical analyses. We then successfully developed solubilization and purification processes for three of these proteins. Finally, we questioned the functionality of a peripheral and an intrinsic membrane protein, and demonstrated that both proteins were active when produced in this system.Conclusions/significanceAltogether, these data suggest that Lactococcus lactis might be an attractive system for the efficient and functional production of difficult plant membrane proteins.
Published: 2010
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10. Optical Spectroscopy Methods to Monitor Cells and Bacteria Concentrations and to Detect Contamination During Cell Culture: Application to the Fabrication of ATMPs.

Author: Bruno Wacogne, Déborah Legrand, Charles-Louis Azzopardi, Christian Pieralli, and Annie Frelet-Barrand
Published: 2020
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11. Optical Spectroscopy for the Quality Control of ATMP Fabrication: A New Method to Monitor Cell Expansion and to Detect Contaminations.

Author: Bruno Wacogne, Déborah Legrand, Christian Pieralli, and Annie Frelet-Barrand
Published: 2020
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12. Online Monitoring of T-Cell Growth: Assessment of the Concentration and Quality, and Real-Time Detection of Contamination

Author: Bruno Wacogne, Naïs Vaccari, Marine Belinger-Podevin, Claudia Koubevi, Alain Rouleau, and Annie Frelet-Barrand
Published: 2022
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13. Optical Spectroscopy Methods to Monitor Cells and Bacteria Concentrations and to Detect Contamination during Cell Culture: Application to the Fabrication of ATMPs

Author: Déborah Legrand, Annie Frelet-Barrand, Bruno Wacogne, Charles-Louis Azzopardi, Christian Pieralli, Franche-Comté Électronique Mécanique, Thermique et Optique - Sciences et Technologies (UMR 6174) (FEMTO-ST), Université de Technologie de Belfort-Montbeliard (UTBM)-Ecole Nationale Supérieure de Mécanique et des Microtechniques (ENSMM)-Université de Franche-Comté (UFC), and Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC)-Centre National de la Recherche Scientifique (CNRS)
Subjects: [SPI.ACOU]Engineering Sciences [physics]/Acoustics [physics.class-ph], business.industry, 010401 analytical chemistry, 02 engineering and technology, Expansion phase, Contamination, 021001 nanoscience & nanotechnology, 01 natural sciences, 0104 chemical sciences, 3. Good health, Highly sensitive, [SPI.MAT]Engineering Sciences [physics]/Materials, Real-time Control System, Bioreactor, Environmental science, [SPI.NANO]Engineering Sciences [physics]/Micro and nanotechnologies/Microelectronics, 0210 nano-technology, Process engineering, business
Abstract: International audience; Currently, the production of Advanced Therapy Medicinal Products is highly sensitive to any contamination sources and therefore takes place in clean and sterile environments. Several days are required for each production, making these products extremely expensive. Throughout the process, numerous quality controls must be performed. This is especially true during the expansion phase in order to monitor cell growth and to detect any contamination. Bioreactor’s content must periodically be sampled to perform these controls. Two major drawbacks can be identified: a delayed knowledge of the quality control result and an additional risk of new contaminations due to sampling. In this work, we present optical spectroscopy methods which can be used to drastically reduce the risk of contamination. They provide a real time control of what happens in the bioreactor in a closed system manner. Cell concentrations are measured with an accuracy below 5% and contamination can be detected about 3 h after it occurred. The real time operation leads to several tens of thousand dollars’ savings because it allows stopping the production as soon as a problem arises. Consequently, the price of these products should be greatly reduced and they may be proposed to more patients.
Published: 2021

14. Optical spectroscopy for the quality control of ATMP fabrication: a new method to monitor cell expansion and to detect contamination

Author: Déborah Legrand, Christian Pieralli, Annie Frelet-Barrand, Bruno Wacogne, Franche-Comté Électronique Mécanique, Thermique et Optique - Sciences et Technologies (UMR 6174) (FEMTO-ST), Université de Technologie de Belfort-Montbeliard (UTBM)-Ecole Nationale Supérieure de Mécanique et des Microtechniques (ENSMM)-Université de Franche-Comté (UFC), and Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Cell expansion, chemistry.chemical_compound, [SPI]Engineering Sciences [physics], Quality (physics), Fabrication, Materials science, chemistry, Nanotechnology, ATMP, Spectroscopy, ComputingMilieux_MISCELLANEOUS
Abstract: International audience
Published: 2020
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15. Ectopic Neo-Formed Intracellular Membranes in Escherichia coli: A Response to Membrane Protein-Induced Stress Involving Membrane Curvature and Domains

Author: Stéphane Orlowski, Nadège Jamin, Christine Jaxel, Manuel Garrigos, Annie Frelet-Barrand, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Laboratoire des Protéines et Systèmes Membranaires (LPSM), Département Biochimie, Biophysique et Biologie Structurale (B3S), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Franche-Comté Électronique Mécanique, Thermique et Optique - Sciences et Technologies (UMR 6174) (FEMTO-ST), Université de Technologie de Belfort-Montbeliard (UTBM)-Ecole Nationale Supérieure de Mécanique et des Microtechniques (ENSMM)-Université de Franche-Comté (UFC), Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC)-Centre National de la Recherche Scientifique (CNRS), Université de Technologie de Belfort-Montbeliard (UTBM)-Ecole Nationale Supérieure de Mécanique et des Microtechniques (ENSMM)-Centre National de la Recherche Scientifique (CNRS)-Université de Franche-Comté (UFC), Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC), and ANR-17-CE11-0015,CAVEOTANK,Production de protéines membranaires recombinantes insérées dans les membranes de cavéoles hétérologues(2017)
Subjects: 0301 basic medicine, lcsh:QR1-502, Review, Biochemistry, lcsh:Microbiology, [SPI.MAT]Engineering Sciences [physics]/Materials, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Protein structure, Membrane Microdomains, Stress, Physiological, Cardiolipin, Escherichia coli, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SPI.NANO]Engineering Sciences [physics]/Micro and nanotechnologies/Microelectronics, Molecular Biology, ComputingMilieux_MISCELLANEOUS, [SPI.ACOU]Engineering Sciences [physics]/Acoustics [physics.class-ph], vesicles, Vesicle, Membrane Proteins, intracellular membranes, membrane protein overexpression, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], 030104 developmental biology, Membrane, Membrane protein, chemistry, membrane domains, Membrane curvature, Cytoplasm, membrane curvature, Biophysics, tubules, 030217 neurology & neurosurgery, Intracellular
Abstract: International audience; Bacterial cytoplasmic membrane stress induced by the overexpression of membrane proteins at high levels can lead to formation of ectopic intracellular membranes. In this review, we report the various observations of such membranes in Escherichia coli, compare their morphological and biochemical characterizations, and we analyze the underlying molecular processes leading to their formation. Actually, these membranes display either vesicular or tubular structures, are separated or connected to the cytoplasmic membrane, present mono- or polydispersed sizes and shapes, and possess ordered or disordered arrangements. Moreover, their composition differs from that of the cytoplasmic membrane, with high amounts of the overexpressed membrane protein and altered lipid-to-protein ratio and cardiolipin content. These data reveal the importance of membrane domains, based on local specific lipid–protein and protein–protein interactions, with both being crucial for local membrane curvature generation, and they highlight the strong influence of protein structure. Indeed, whether the cylindrically or spherically curvature-active proteins are actively curvogenic or passively curvophilic, the underlying molecular scenarios are different and can be correlated with the morphological features of the neo-formed internal membranes. Delineating these molecular mechanisms is highly desirable for a better understanding of protein–lipid interactions within membrane domains, and for optimization of high-level membrane protein production in E. coli.
Published: 2018
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16. The NOS-like protein from the microalgae Ostreococcus tauri is a genuine and ultrafast NO-producing enzyme

Author: Pierre Dorlet, Annie Frelet-Barrand, Jérôme Santolini, Lorenzo Lamattina, Marine Weisslocker-Schaetzel, Mehdi Lembrouk, Noelia Foresi, François André, Nabila Touazi, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Stress Oxydants et Détoxication (LSOD), Département Biochimie, Biophysique et Biologie Structurale (B3S), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Franche-Comté Électronique Mécanique, Thermique et Optique - Sciences et Technologies (UMR 6174) (FEMTO-ST), Université de Technologie de Belfort-Montbeliard (UTBM)-Ecole Nationale Supérieure de Mécanique et des Microtechniques (ENSMM)-Centre National de la Recherche Scientifique (CNRS)-Université de Franche-Comté (UFC), Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC), Instituto de Investigaciones Biológicas [Mar del Plata] (IIB), Consejo Nacional de Investigaciones Científicas y Técnicas [Buenos Aires] (CONICET)-Facultad de Ciencias Exactas y Naturales [Mar del Plata], and Universidad Nacional de Mar del Plata [Mar del Plata] (UNMdP)-Universidad Nacional de Mar del Plata [Mar del Plata] (UNMdP)
Subjects: 0301 basic medicine, STRUCTURE, Algae, Lineage (evolution), Otras Ciencias Biológicas, [SDV]Life Sciences [q-bio], FUNCTION, Sequence alignment, NO SYNTHASE, Plant Science, Computational biology, Biology, Genome, Catalysis, Ostreococcus tauri, [SPI.MAT]Engineering Sciences [physics]/Materials, Ciencias Biológicas, 03 medical and health sciences, ALGAE, NO synthase, Phylogenetics, Chlorophyta, Genetics, Microalgae, Homology modeling, Amino Acid Sequence, PLANT, Function, [SPI.NANO]Engineering Sciences [physics]/Micro and nanotechnologies/Microelectronics, Phylogeny, [SPI.ACOU]Engineering Sciences [physics]/Acoustics [physics.class-ph], 030102 biochemistry & molecular biology, Phylogenetic tree, CATALYSIS, Algal Proteins, Structure, General Medicine, Plant, biology.organism_classification, 030104 developmental biology, Biochemistry, Nitric Oxide Synthase, Agronomy and Crop Science, Sequence Alignment, Function (biology), CIENCIAS NATURALES Y EXACTAS
Abstract: The exponential increase of genomes’ sequencing has revealed the presence of NO-Synthases (NOS) throughout the tree of life, uncovering an extraordinary diversity of genetic structure and biological functions. Although NO has been shown to be a crucial mediator in plant physiology, NOS sequences seem present solely in green algae genomes, with a first identification in the picoplankton species Ostreococcus tauri. There is no rationale so far to account for the presence of NOS in this early-diverging branch of the green lineage and its absence in land plants. To address the biological function of algae NOS, we cloned, expressed and characterized the NOS oxygenase domain from Ostreococcus tauri (OtNOSoxy). We launched a phylogenetic and structural analysis of algae NOS, and achieved a 3D model of OtNOSoxy by homology modeling. We used a combination of various spectroscopies to characterize the structural and electronic fingerprints of some OtNOSoxy reaction intermediates. The analysis of OtNOSoxy catalytic activity and kinetic efficiency was achieved by stoichiometric stopped-flow. Our results highlight the conserved and particular features of OtNOSoxy structure that might explain its ultrafast NO-producing capacity. This integrative Structure-Catalysis-Function approach could be extended to the whole NOS superfamily and used for predicting potential biological activity for any new NOS. Fil: Weisslocker-Schaetzel, Marine. Université Paris Sud; Francia Fil: André, François. Université Paris Sud; Francia Fil: Touazi, Nabila. Université Paris Sud; Francia Fil: Foresi, Noelia Pamela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Lembrouk, Mehdi. Université Paris Sud; Francia Fil: Dorlet, Pierre. Université Paris Sud; Francia Fil: Frelet Barrand, Annie. Université Paris Sud; Francia Fil: Lamattina, Lorenzo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Santolini, Jérôme. Université Paris Sud; Francia
Published: 2017
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17. Membrane Protein Production in Lactococcus lactis for Functional Studies

Author: Daphne, Seigneurin-Berny, Martin S, King, Emiline, Sautron, Lucas, Moyet, Patrice, Catty, François, André, Norbert, Rolland, Edmund R S, Kunji, and Annie, Frelet-Barrand
Subjects: Lactococcus lactis, Bacterial Proteins, Genetic Vectors, Gene Expression, Membrane Proteins, Protein Engineering, Recombinant Proteins
Abstract: Due to their unique properties, expression and study of membrane proteins in heterologous systems remains difficult. Among the bacterial systems available, the Gram-positive lactic bacterium, Lactococcus lactis, traditionally used in food fermentations, is nowadays widely used for large-scale production and functional characterization of bacterial and eukaryotic membrane proteins. The aim of this chapter is to describe the different possibilities for the functional characterization of peripheral or intrinsic membrane proteins expressed in Lactococcus lactis.
Published: 2016

18. Lactococcus lactis is an Efficient Expression System for Mammalian Membrane Proteins Involved in Liver Detoxification, CYP3A4, and MGST1

Author: Stéphane Orlowski, Sana Bakari, Marcel Delaforge, Laura Sourd, Mehdi Lembrouk, Annie Frelet-Barrand, Farès Ousalem, François André, Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), FEMTO-ST Institute, Université de Franche-Comté ( UFC ) -CNRS : UMR6174-Université de Technologie de Belfort-Montbeliard, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Franche-Comté Électronique Mécanique, Thermique et Optique - Sciences et Technologies (UMR 6174) (FEMTO-ST), Université de Technologie de Belfort-Montbeliard (UTBM)-Ecole Nationale Supérieure de Mécanique et des Microtechniques (ENSMM)-Université de Franche-Comté (UFC), Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC)-Centre National de la Recherche Scientifique (CNRS), Université de Technologie de Belfort-Montbeliard (UTBM)-Ecole Nationale Supérieure de Mécanique et des Microtechniques (ENSMM)-Centre National de la Recherche Scientifique (CNRS)-Université de Franche-Comté (UFC), and Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC)
Subjects: 0301 basic medicine, [SDV]Life Sciences [q-bio], Saccharomyces cerevisiae, Gene Expression, Bioengineering, Cytochrome P450, medicine.disease_cause, Applied Microbiology and Biotechnology, Biochemistry, [SPI.MAT]Engineering Sciences [physics]/Materials, 03 medical and health sciences, Membrane proteins, medicine, Animals, Cytochrome P-450 CYP3A, Humans, Cloning, Molecular, [SPI.NANO]Engineering Sciences [physics]/Micro and nanotechnologies/Microelectronics, Molecular Biology, Gene, Escherichia coli, Glutathione Transferase, Cloning, [SPI.ACOU]Engineering Sciences [physics]/Acoustics [physics.class-ph], 030102 biochemistry & molecular biology, biology, [ SDV ] Life Sciences [q-bio], Lactococcus lactis, biology.organism_classification, Recombinant Proteins, Rats, 030104 developmental biology, Liver, Membrane protein, Microsomal glutathione S-transferase, biology.protein, Heterologous expression, Biotechnology, Gateway
Abstract: International audience; Despite the great importance of human membrane proteins involved in detoxification mechanisms, their wide use for biochemical approaches is still hampered by several technical difficulties considering eukaryotic protein expression in order to obtain the large amounts of protein required for functional and/or structural studies. Lactococcus lactis has emerged recently as an alternative heterologous expression system to Escherichia coli for proteins that are difficult to express. The aim of this work was to check its ability to express mammalian membrane proteins involved in liver detoxification, i.e., CYP3A4 and two isoforms of MGST1 (rat and human). Genes were cloned using two different strategies, i.e., classical or Gateway-compatible cloning, and we checked the possible influence of two affinity tags (6×-His-tag and Strep-tag II). Interestingly, all proteins could be successfully expressed in L. lactis at higher yields than those previously obtained for these proteins with classical expression systems (E. coli, Saccharomyces cerevisiae) or those of other eukaryotic membrane proteins expressed in L. lactis. In addition, rMGST1 was fairly active after expression in L. lactis. This study highlights L. lactis as an attractive system for efficient expression of mammalian detoxification membrane proteins at levels compatible with further functional and structural studies.
Published: 2016
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19. The Arabidopsis ATP-binding Cassette Protein AtMRP5/AtABCC5 Is a High Affinity Inositol Hexakisphosphate Transporter Involved in Guard Cell Signaling and Phytate Storage

Author: Barbara Weder, Porntip Green, Markus Klein, Annie Frelet-Barrand, Enrico Martinoia, Charles A. Brearley, Jan K. Schjoerring, Réka Nagy, and Hanne Grob
Subjects: Phytic Acid, Inositol Phosphates, Adenylyl Imidodiphosphate, Green Fluorescent Proteins, Mutant, ATP-binding cassette transporter, Biology, Biochemistry, Plant Epidermis, chemistry.chemical_compound, Microsomes, Yeasts, Arabidopsis, Guard cell, Inositol, Molecular Biology, Microscopy, Confocal, Arabidopsis Proteins, Genetic Complementation Test, Biological Transport, Phosphorus, Cell Biology, Plants, Genetically Modified, biology.organism_classification, Complementation, Membrane Transport, Structure, Function, and Biogenesis, chemistry, Mutation, Plant Stomata, Seeds, Multidrug Resistance-Associated Proteins, Signal transduction, Signal Transduction
Abstract: Arabidopsis possesses a superfamily of ATP-binding cassette (ABC) transporters. Among these, the multidrug resistance-associated protein AtMRP5/AtABCC5 regulates stomatal aperture and controls plasma membrane anion channels of guard cells. Remarkably, despite the prominent role of AtMRP5 in conferring partial drought insensitivity upon Arabidopsis, we know little of the biochemical function of AtMRP5. Our phylogenetic analysis showed that AtMRP5 is closely related to maize MRP4, mutation of which confers a low inositol hexakisphosphate kernel phenotype. We now show that insertion mutants of AtMRP5 display a low inositol hexakisphosphate phenotype in seed tissue and that this phenotype is associated with alterations of mineral cation and phosphate status. By heterologous expression in yeast, we demonstrate that AtMRP5 encodes a specific and high affinity ATP-dependent inositol hexakisphosphate transporter that is sensitive to inhibitors of ABC transporters. Moreover, complementation of the mrp5-1 insertion mutants of Arabidopsis with the AtMRP5 cDNA driven from a guard cell-specific promoter restores the sensitivity of the mutant to abscisic acid-mediated inhibition of stomatal opening. Additionally, we show that mutation of residues of the Walker B motif prevents restoring the multiple phenotypes associated with mrp5-1. Our findings highlight a novel function of plant ABC transporters that may be relevant to other kingdoms. They also extend the signaling repertoire of this ubiquitous inositol polyphosphate signaling molecule.
Published: 2009
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20. Membrane Protein Production in Lactococcus lactis for Functional Studies

Author: Lucas Moyet, Edmund R.S. Kunji, Martin S. King, Annie Frelet-Barrand, Emiline Sautron, Patrice Catty, François André, Daphné Seigneurin-Berny, Norbert Rolland, Physiologie cellulaire et végétale (LPCV), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-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), Mitochondrial Biology Unit, Medical Research Council, Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Franche-Comté Électronique Mécanique, Thermique et Optique - Sciences et Technologies (UMR 6174) (FEMTO-ST), Université de Technologie de Belfort-Montbeliard (UTBM)-Ecole Nationale Supérieure de Mécanique et des Microtechniques (ENSMM)-Université de Franche-Comté (UFC), Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC)-Centre National de la Recherche Scientifique (CNRS), Medical Research Council [MC_U105663139], Mus-Veteau Isabelle, Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Université de Franche-Comté (UFC)-Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Supérieure de Mécanique et des Microtechniques (ENSMM)-Université de Technologie de Belfort-Montbeliard (UTBM), Laboratoire de physiologie cellulaire végétale ( LPCV ), Institut National de la Recherche Agronomique ( INRA ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Grenoble Alpes ( UGA ), Laboratoire de Chimie et Biologie des Métaux ( LCBM - UMR 5249 ), Université Joseph Fourier - Grenoble 1 ( UJF ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Grenoble Alpes ( UGA ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ), Mus-Veteau, I., Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge [UK] (CAM), Université de Technologie de Belfort-Montbeliard (UTBM)-Ecole Nationale Supérieure de Mécanique et des Microtechniques (ENSMM)-Centre National de la Recherche Scientifique (CNRS)-Université de Franche-Comté (UFC), Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC), and ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010)
Subjects: 0301 basic medicine, Heterologous, Expression, law.invention, 03 medical and health sciences, Transport assays, law, Gene expression, Membrane proteins, Protocol, Functional studies, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], [ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030102 biochemistry & molecular biology, biology, [ SDV ] Life Sciences [q-bio], Lactococcus lactis, food and beverages, Protein engineering, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], 030104 developmental biology, Biochemistry, Membrane protein, Recombinant DNA, Protein overexpression, Bacteria
Abstract: International audience; Due to their unique properties, expression and study of membrane proteins in heterologous systems remains difficult. Among the bacterial systems available, the Gram-positive lactic bacterium, Lactococcus lactis, traditionally used in food fermentations, is nowadays widely used for large-scale production and functional characterization of bacterial and eukaryotic membrane proteins. The aim of this chapter is to describe the different possibilities for the functional characterization of peripheral or intrinsic membrane proteins expressed in Lactococcus lactis.
Published: 2016
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21. Functional Expression of Plant Membrane Proteins in Lactococcus lactis

Author: Annie Frelet-Barrand, Daniel Salvi, Lucas Moyet, Emeline Sautron, Sylvain Boutigny, Daphné Seigneurin-Berny, Norbert Rolland, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Stress Oxydants et Détoxication (LSOD), Département Biochimie, Biophysique et Biologie Structurale (B3S), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), García-Fruitós, Elena, Laboratoire de physiologie cellulaire végétale (LPCV), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), E, Garcia-Fruitos, Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-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)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Subjects: biology, Chemistry, Lactococcus, [SDV]Life Sciences [q-bio], Lactococcus lactis, Heterologous, food and beverages, biology.organism_classification, protein overexpression, Membrane, Biochemistry, Membrane protein, Solubilization, Functional expression, Protocol, membrane protein, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Lactococcus lacti, Heterologous protein, bacteria, Bacteria, ComputingMilieux_MISCELLANEOUS
Abstract: International audience; The study of most membrane proteins remains challenging due to their hydrophobicity and their low natural abundance in cells. Lactococcus lactis, a Gram-positive lactic bacterium, has been traditionally used in food fermentations and is nowadays widely used in biotechnology for large-scale production of heterologous proteins. This system has been successfully used for the production of prokaryotic and eukaryotic membrane proteins. The purpose of this chapter is to provide detailed protocols for (1) the expression of plant peripheral or intrinsic membrane proteins and then for (2) their solubilization, from Lactococcus membranes, for further purification steps and biochemical characterization.
Published: 2015
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22. Functional expression of plant membrane proteins in Lactococcus lactis

Author: Sylvain, Boutigny, Emeline, Sautron, Annie, Frelet-Barrand, Lucas, Moyet, Daniel, Salvi, Norbert, Rolland, and Daphné, Seigneurin-Berny
Subjects: Lactococcus lactis, Gene Expression, Membrane Proteins, Biotechnology, Plant Proteins
Abstract: The study of most membrane proteins remains challenging due to their hydrophobicity and their low natural abundance in cells. Lactococcus lactis, a Gram-positive lactic bacterium, has been traditionally used in food fermentations and is nowadays widely used in biotechnology for large-scale production of heterologous proteins. This system has been successfully used for the production of prokaryotic and eukaryotic membrane proteins. The purpose of this chapter is to provide detailed protocols for (1) the expression of plant peripheral or intrinsic membrane proteins and then for (2) their solubilization, from Lactococcus membranes, for further purification steps and biochemical characterization.
Published: 2014

23. HMA1 and PAA1, two chloroplast envelope PIB-ATPases, play distinct roles in chloroplast copper homeostasis

Author: Annie Frelet-Barrand, Giovanni Finazzi, Norbert Rolland, Corinne Rivasseau, Daphné Seigneurin-Berny, Marinus Pilon, Emeline Sautron, Sylvain Boutigny, Laboratoire de physiologie cellulaire végétale (LPCV), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), Biology department and program in molecular plant biology, University of Colorado [Boulder], Grant from the US National Science Foundation (IOS-0847442), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-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), and Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0106 biological sciences, Arabidopsis thaliana, Physiology, ATPase, Mutant, Arabidopsis, chemistry.chemical_element, Gene Expression, plant, Plant Science, 01 natural sciences, Chloroplast membrane, Gene Expression Regulation, Enzymologic, PIB ATPase, 03 medical and health sciences, chloroplast, Gene Expression Regulation, Plant, medicine, Chloroplast Proton-Translocating ATPases, Homeostasis, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Cell damage, 030304 developmental biology, Adenosine Triphosphatases, 0303 health sciences, biology, Arabidopsis Proteins, envelope transporter, food and beverages, Transporter, biology.organism_classification, medicine.disease, Plants, Genetically Modified, Copper, Chloroplast, Plant Leaves, Phenotype, Biochemistry, chemistry, Seedlings, copper, Mutation, biology.protein, metal homeostasis, 010606 plant biology & botany
Abstract: International audience; Copper is an essential micronutrient but it is also potentially toxic as copper ions can catalyse the production of free radicals, which result in various types of cell damage. Therefore, copper homeostasis in plant and animal cells must be tightly controlled. In the chloroplast, copper import is mediated by a chloroplast-envelope PIB-type ATPase, HMA6/PAA1. Copper may also be imported by HMA1, another chloroplast-envelope PIB-ATPase. To get more insights into the specific functional roles of HMA1 and PAA1 in copper homeostasis, this study analysed the phenotypes of plants affected in the expression of both HMA1 and PAA1 ATPases, as well as of plants overexpressing HMA1 in a paa1 mutant background. The results presented here provide new evidence associating HMA1 with copper homeostasis in the chloroplast. These data suggest that HMA1 and PAA1 behave as distinct pathways for copper import and targeting to the chloroplast. Finally, this work also provides evidence for an alternative route for copper import into the chloroplast mediated by an as-yet unidentified transporter that is neither HMA1 nor PAA1
Published: 2014
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24. Lactococcus lactis: Recent Developments in Functional Expression of Membrane Proteins

Author: Marcel Delaforge, Daphné Seigneurin-Berny, François Andre, Norbert Rolland, Annie Frelet-Barrand, and Sana Bakari
Subjects: Membrane protein, Functional expression, Gene expression, Lactococcus lactis, medicine, Prokaryotic expression, A protein, Computational biology, Biology, medicine.disease_cause, biology.organism_classification, Escherichia coli, Microbiology
Abstract: Membrane proteins (MPs) play key roles in most important cellular processes ranging from cell-to-cell communication to signaling processes. Despite recent improvements, the expression of functionally folded MPs in sufficient amounts for functional and structural characterization remains a challenge. Indeed, it is still difficult to predict whether a protein can be overproduced in a functional state in some expression system(s), although studies of high-throughput screens have been issued in recent years. Prokaryotic expression systems present several advantages over eukaryotic ones. Among them, Lactococcus lactis (L. lactis) has emerged in the past years as a good alternative expression system to Escherichia coli. The purpose of this chapter is to describe L. lactis and its tightly inducible system, nisin-controlled gene expression (NICE), for the expression of MPs from both prokaryotic and eukaryotic origins. Moreover, we will describe the functional characterization of some MPs produced in L. lactis.
Published: 2014
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25. Expression of a chloroplast ATP/ADP transporter in E. coli membranes: behind the Mistic strategy

Author: Thierry Vernet, Céline Juillan-Binard, Annie Frelet-Barrand, Norbert Rolland, Aurélien Deniaud, Eva Pebay-Peyroula, Florent Bernaudat, Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-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), Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut de Biosciences et de Biotechnologies de Grenoble (ex-IRTSV) (BIG), 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]), CEA (CEA-PM program) - CNRS (post-doctoral fellowship toAFB) - Université JosephFourierGrenoble 1 - Institut Universitaire de France - Association Française contre les Myopathies (post-doctoral fellowship to AD), Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), 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), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), 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), and 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])
Subjects: Protein Folding, Chloroplasts, MESH: Protein Structure, Quaternary, Arabidopsis, Biochemistry, Protein structure, MESH: Structure-Activity Relationship, Adenosine Triphosphate, MESH: Adenosine Triphosphate, MESH: Amphibian Proteins, membrane protein, MESH: Arabidopsis, Cloning, Molecular, 0303 health sciences, Mistic fusion strategy, MESH: Kinetics, Eukaryote, MESH: Escherichia coli, 030302 biochemistry & molecular biology, heterologous expression, Cell biology, Chloroplast, Adenosine Diphosphate, Nucleotide Transport Proteins, Protein folding, ATP/ADP chloroplast transporter, ATP–ADP translocase, in vivo fusion cleavage, MESH: Nucleotide Transport Proteins, MESH: Peptide Hydrolases, MESH: Protein Folding, MESH: Biological Transport, Recombinant Fusion Proteins, Biophysics, MESH: Arabidopsis Proteins, Biology, Amphibian Proteins, 03 medical and health sciences, Structure-Activity Relationship, Membrane protein heterologous expression, MESH: Recombinant Fusion Proteins, Escherichia coli, MESH: Cloning, Molecular, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Protein Structure, Quaternary, 030304 developmental biology, MESH: Chloroplasts, MESH: Adenosine Diphosphate, Arabidopsis Proteins, Cell Membrane, Transporter, Biological Transport, Cell Biology, biology.organism_classification, Kinetics, Membrane protein, Heterologous expression, MESH: Cell Membrane, Peptide Hydrolases
Abstract: International audience; Eukaryotic membrane protein expression is still a major bottleneck for structural studies. Production in E. coli often leads to low expression level and/or aggregated proteins. In the last decade, strategies relying on new fusion protein expression revealed promising results. Fusion with the amphipatic Mistic protein has been described to favor expression in E. coli membranes. Although, this approach has already been reported for a few membrane proteins, little is known about the activity of the fused proteins. We used this strategy and obtained high expression levels of a chloroplast ATP/ADP transporter from A. thaliana (NTT1) and characterized its transport properties. NTT1 fused to Mistic has a very low transport activity which can be recovered after in vivo Mistic fusion cleavage. Moreover, detailed molecular characterization of purified NTT1 mature form, NTT1 fused to Mistic or NTT1 cleaved-off from this fusion highlights the correct fold of the latter one. Therefore, considering the higher quantity of purified NTT1 mature form obtained via the Mistic fusion approach, this is a valuable strategy for obtaining quantities of pure and active proteins that are adequate for structural studies.
Published: 2011
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26. Heterologous expression of membrane proteins: choosing the appropriate host

Author: Sylvain Boutigny, Elisabeth Darrouzet, Daniel Salvi, Daphné Seigneurin-Berny, Sébastien Dementin, Annie Frelet-Barrand, Thierry Desnos, Eva Pebay-Peyroula, Norbert Rolland, Monique Sabaty, Jean-Baptiste Rioux, Florent Bernaudat, Jacques Joyard, Patrick Hivin, Nathalie Pochon, David Pignol, Pierre Richaud, Thierry Vernet, Institut de biologie structurale (IBS - UMR 5075), 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)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Signalisation de l'Adaptation des Végétaux à l'Environnement (SAVE), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), 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)-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), Microbiologie Environnementale et Moléculaire (MEM), Laboratoire d'Ingénierie Cellulaire et de Biologie (LICB), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire des Echanges Membranaires et Signalisation (LEMS), Université de la Méditerranée - Aix-Marseille 2-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Transporteurs en Imagerie et Radiothérapie en Oncologie (TIRO - UMR E4320), Service Hospitalier Frédéric Joliot (SHFJ), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-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)-Université Paris-Saclay-UMR E4320 (TIRO-MATOs), Université Nice Sophia Antipolis (... - 2019) (UNS), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Nice Sophia Antipolis (... - 2019) (UNS), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Nice Sophia Antipolis (... - 2019) (UNS), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), 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), 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), Transporteurs et Imagerie, Radiothérapie en Oncologie et Mécanismes biologiques des Altérations du Tissu Osseux (TIRO-MATOs - UMR E4320), UMR E4320 (TIRO-MATOs), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA)-Service Hospitalier Frédéric Joliot (SHFJ), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, This work has been supported by the CEA (Commissariat a l'Energie Atomique et aux Energies Alternatives, funding of the CEA-PM project and PostDoc fellowships to F. Bernaudat, A. Frelet-Barrand, P. Hivin and S. Dementin), by the CNRS (Centre National de la Recherche Scientifique, Post-Doc fellowship to A. Frelet-Barrand) and by the University of Grenoble 1. This work was partly supported by the European grant LSMH-CT-EUR-INTAFAR 2004-512138. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Plant Environmental Physiology and Stress Signaling (PEPSS), Université Paris-Saclay-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)-UMR E4320 (TIRO-MATOs), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA), and Bernaudat, Florent
Subjects: Arabidopsis thaliana, Gene Expression, plant, Plant Science, medicine.disease_cause, Bioinformatics, Polymerase Chain Reaction, Biochemistry, law.invention, expression system, Transmembrane Transport Proteins, law, Molecular Cell Biology, Cloning, Molecular, Baculovirus, bacteria, membrane, Cellular proteins, Plant Proteins, bactérie, 0303 health sciences, Multidisciplinary, biology, Genetically Modified Organisms, Recombinant Proteins, Cell biology, Blot, Lactococcus lactis, expression host, Recombinant DNA, Cytochemistry, Medicine, bacterial proteins, dna primers, membrane proteins, plant proteins, multidisciplinary sciences, science and technology, Research Article, Biotechnology, Science, Blotting, Western, Rhodobacter sphaeroides, Spodoptera, Cell Line, 03 medical and health sciences, Bacterial Proteins, medicine, Escherichia coli, Nicotiana benthamiana, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Biology, Transgenic Plants, 030304 developmental biology, DNA Primers, Cloning, Base Sequence, 030306 microbiology, Cell Membrane, Membrane Proteins, Proteins, insecta, biology.organism_classification, Transmembrane Proteins, Membrane protein, Plant Biotechnology, Heterologous expression, protein, mamalia, overexpression
Abstract: International audience; 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.
Published: 2011
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27. High-chloride concentrations abolish the binding of adenine nucleotides in the mitochondrial ADP/ATP carrier family

Author: Christophe Chipot, François Dehez, Stéphanie Ravaud, Eva Pebay-Peyroula, Eva-Maria Krammer, Annie Frelet-Barrand, Structure et Réactivité des Systèmes Moléculaires Complexes (SRSMC), Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075), 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)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie du CNRS (INC)-Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), and 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)
Subjects: Models, Molecular, Time Factors, Protein Conformation, Arabidopsis, MESH: Adenine Nucleotides, MESH: Amino Acid Sequence, Sodium Chloride, 01 natural sciences, MESH: Electromagnetic Fields, MESH: Recombinant Proteins, chemistry.chemical_compound, Adenosine Triphosphate, Protein structure, MESH: Protein Conformation, Models, Adenine nucleotide, MESH: Adenosine Triphosphate, MESH: Animals, MESH: Arabidopsis, Inner mitochondrial membrane, Conserved Sequence, MESH: Static Electricity, chemistry.chemical_classification, 0303 health sciences, MESH: Conserved Sequence, Adenine Nucleotides, MESH: Escherichia coli, Recombinant Proteins, Amino acid, MESH: Cattle, Biochemistry, ATP–ADP translocase, MESH: Mitochondrial ADP, ATP Translocases, MESH: Models, Molecular, MESH: Sodium Chloride, Static Electricity, Biophysics, MESH: Arabidopsis Proteins, Biology, 010402 general chemistry, 03 medical and health sciences, Electromagnetic Fields, Chlorides, MESH: Computer Simulation, Escherichia coli, Animals, Mitochondrial ADP, Computer Simulation, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, ATP Translocases, Binding site, MESH: Chlorides, 030304 developmental biology, Biophysical Letter, Arabidopsis Proteins, MESH: Time Factors, Molecular, Mitochondrial carrier, 0104 chemical sciences, chemistry, Cattle, Mitochondrial ADP, ATP Translocases, Adenosine triphosphate
Abstract: International audience; The ADP/ATP carrier (AAC) is a very effective membrane protein that mediates the exchange of ADP and ATP across the mitochondrial membrane. In vivo transport measurements on the AAC overexpressed in Escherichia coli demonstrate that this process can be severely inhibited by high-chloride concentrations. Molecular-dynamics simulations reveal a strong modification of the topology of the local electric field related to the number of chloride ions inside the cavity. Halide ions are shown to shield the positive charges lining the internal cavity of the carrier by accurate targeting of key basic residues. These specific amino acids are highly conserved as highlighted by the analysis of multiple AAC sequences. These results strongly suggest that the chloride concentration acts as an electrostatic lock for the mitochondrial AAC family, thereby preventing adenine nucleotides from reaching their dedicated binding sites.
Published: 2009
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28. Membrane Protein Expression in Lactococcus lactis

Author: Annie Frelet-Barrand, Norbert Rolland, Edmund R.S. Kunji, and Sylvain Boutigny
Subjects: 0303 health sciences, Vesicle-associated membrane protein 8, Expression vector, biology, 030306 microbiology, Lactococcus lactis, biology.organism_classification, 03 medical and health sciences, Protein structure, Membrane protein, Biochemistry, Gene expression, Gene, Integral membrane protein, 030304 developmental biology
Abstract: Membrane proteins play key roles in cellular physiology, and they are important drug targets. Approximately 25% of all genes identified in sequenced genomes are known to encode membrane proteins; however, the majority have no assigned function. Although the resolution of soluble protein structure has entered the high-throughput stage, only 100 high-resolution structures of membrane proteins have been described until now. Lactococcus lactis is a gram-positive lactic bacterium that has been used traditionally in food fermentations, but it is now used widely in biotechnology for large-scale overproduction of heterologously expressed proteins. Various expression vectors based on either constitutive or inducible promoters exist. The nisin-inducible controlled gene expression (NICE) system is the most suitable for recombinant membrane protein expression allowing for fine control of gene expression based on the autoregulation mechanism of the bacteriocin nisin. Recombinant membrane proteins can be produced with affinity tags for efficient detection and purification from crude membrane protein extracts. The purpose of this chapter is to provide a detailed protocol for the expression of membrane proteins and their detection using the Strep-tag II affinity tag in L. lactis.
Published: 2009
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29. Lactococcus lactis, an alternative system for functional expression of peripheral and intrinsic Arabidopsis membrane proteins

Author: Lucas Moyet, Norbert Rolland, Jacques Joyard, Annie Frelet-Barrand, Aurélien Deniaud, Sylvain Boutigny, Daniel Salvi, Pierre Richaud, Daphné Seigneurin-Berny, Eva Pebay-Peyroula, Florent Bernaudat, Laboratoire de physiologie cellulaire végétale (LPCV), 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), Institut de biologie structurale (IBS - UMR 5075 ), 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)-Centre National de la Recherche Scientifique (CNRS), Laboratoire des Echanges Membranaires et Signalisation (LEMS), 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 Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)
Subjects: DNA, Complementary, Arabidopsis thaliana, Biochemistry/Membrane Proteins and Energy Transduction, Science, Genetic Vectors, Molecular Sequence Data, plant, membrane protein, expression system, recombinant protein, bacteria, Lactococcus lactis, Heterologous, Plant Biology/Plant Biochemistry and Physiology, 03 medical and health sciences, Open Reading Frames, Arabidopsis, Gene expression, SDV:BBM, Biotechnology/Applied Microbiology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, 030304 developmental biology, Genetics, 0303 health sciences, Multidisciplinary, Expression vector, biology, Base Sequence, 030306 microbiology, cDNA library, Arabidopsis Proteins, biology.organism_classification, Biochemistry, Membrane protein, Medicine, Electrophoresis, Polyacrylamide Gel, Heterologous expression, Biotechnology/Bioengineering, Research Article
Abstract: International audience; BACKGROUND: Despite their functional and biotechnological importance, the study of membrane proteins remains difficult due to their hydrophobicity and their low natural abundance in cells. Furthermore, into established heterologous systems, these proteins are frequently only produced at very low levels, toxic and mis- or unfolded. Lactococcus lactis, a gram-positive lactic bacterium, has been traditionally used in food fermentations. This expression system is also widely used in biotechnology for large-scale production of heterologous proteins. Various expression vectors, based either on constitutive or inducible promoters, are available for this system. While previously used to produce bacterial and eukaryotic membrane proteins, the ability of this system to produce plant membrane proteins was until now not tested. METHODOLOGY/PRINCIPAL FINDINGS: The aim of this work was to test the expression, in Lactococcus lactis, of either peripheral or intrinsic Arabidopsis membrane proteins that could not be produced, or in too low amount, using more classical heterologous expression systems. In an effort to easily transfer genes from Gateway-based Arabidopsis cDNA libraries to the L. lactis expression vector pNZ8148, we first established a cloning strategy compatible with Gateway entry vectors. Interestingly, the six tested Arabidopsis membrane proteins could be produced, in Lactococcus lactis, at levels compatible with further biochemical analyses. We then successfully developed solubilization and purification processes for three of these proteins. Finally, we questioned the functionality of a peripheral and an intrinsic membrane protein, and demonstrated that both proteins were active when produced in this system. CONCLUSIONS/SIGNIFICANCE: Altogether, these data suggest that Lactococcus lactis might be an attractive system for the efficient and functional production of difficult plant membrane proteins.
Published: 2009
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30. Comparative mutant analysis of Arabidopsis ABCC-type ABC transporters: AtMRP2 contributes to detoxification, vacuolar organic anion transport and chlorophyll degradation

Author: Markus Klein, Maika Rüffer, Annie Frelet-Barrand, H. Üner Kolukisaoglu, Sonia Plaza, Burkhard Schulz, Stefan Hörtensteiner, Krasimira Marinova, Louis Azevedo, Barbara Weder, University of Zurich, and Klein, M
Subjects: Chlorophyll, DNA, Bacterial, Organic anion transporter 1, Physiology, Mutant, DNA Mutational Analysis, Arabidopsis, Organic Anion Transporters, ATP-binding cassette transporter, Plant Science, Flowers, Biology, 580 Plants (Botany), Genes, Plant, Plant Roots, 1307 Cell Biology, 10126 Department of Plant and Microbial Biology, Gene Expression Regulation, Plant, Toxicity Tests, 1110 Plant Science, Arabidopsis thaliana, Promoter Regions, Genetic, Alleles, Arabidopsis Proteins, Biological Transport, Cell Biology, General Medicine, 1314 Physiology, biology.organism_classification, Biochemistry, Vacuolar transport, Inactivation, Metabolic, Mutation, Vacuoles, Organic anion transport, biology.protein, ATP-Binding Cassette Transporters, Heterologous expression, Multidrug Resistance-Associated Proteins, Cotyledon, Glucosidases
Abstract: The enormous metabolic plasticity of plants allows detoxification of many harmful compounds that are generated during biosynthetic processes or are present as biotic or abiotic toxins in their environment. Derivatives of toxic compounds such as glutathione conjugates are moved into the central vacuole via ATP-binding cassette (ABC)-type transporters of the multidrug resistance-associated protein (MRP) subfamily. The Arabidopsis genome contains 15 AtMRP isogenes, four of which (AtMRP1, 2, 11 and 12) cluster together in one of two major phylogenetic clades. We isolated T-DNA knockout alleles in all four highly homologous AtMRP genes of this clade and subjected them to physiological analysis to assess the function of each AtMRP of this group. None of the single atmrp mutants displayed visible phenotypes under control conditions. In spite of the fact that AtMRP1 and AtMRP2 had been described as efficient ATP-dependent organic anion transporters in heterologous expression experiments, the contribution of three of the AtMRP genes (1, 11 and 12) to detoxification is marginal. Only knockouts in AtMRP2 exhibited a reduced sensitivity towards 1-chloro-2,4-dinitrobenzene, but not towards other herbicides. AtMRP2 but not AtMRP1, 11 and 12 is involved in chlorophyll degradation since ethylene-treated rosettes of atmrp2 showed reduced senescence, and AtMRP2 expression is induced during senescence. This suggests that AtMRP2 is involved in vacuolar transport of chlorophyll catabolites. Vacuolar uptake studies demonstrated that transport of typical MRP substrates was reduced in atmrp2. We conclude that within clade I, only AtMRP2 contributes significantly to overall organic anion pump activity in vivo.
Published: 2008

31. Comparative Mutant Analysis of Arabidopsis ABCC-Type ABC Transporters: AtMRP2 Contributes to Detoxification, Vacuolar Organic Anion Transport and Chlorophyll Degradation.

Author: Annie Frelet-Barrand, H. Üner Kolukisaoglu, Sonia Plaza, Maika Rüffer, Louis Azevedo, Stefan Hörtensteiner, Krasimira Marinova, Barbara Weder, Burkhard Schulz, and Markus Klein
Subjects: *GENES, *DETOXIFICATION (Alternative medicine), *AGING, *GENETICS
Abstract: The enormous metabolic plasticity of plants allows detoxification of many harmful compounds that are generated during biosynthetic processes or are present as biotic or abiotic toxins in their environment. Derivatives of toxic compounds such as glutathione conjugates are moved into the central vacuole via ATP-binding cassette (ABC)-type transporters of the multidrug resistance-associated protein (MRP) subfamily. The Arabidopsis genome contains 15 AtMRP isogenes, four of which (AtMRP1, 2, 11 and 12) cluster together in one of two major phylogenetic clades. We isolated T-DNA knockout alleles in all four highly homologous AtMRP genes of this clade and subjected them to physiological analysis to assess the function of each AtMRP of this group. None of the single atmrp mutants displayed visible phenotypes under control conditions. In spite of the fact that AtMRP1 and AtMRP2 had been described as efficient ATP-dependent organic anion transporters in heterologous expression experiments, the contribution of three of the AtMRP genes (1, 11 and 12) to detoxification is marginal. Only knockouts in AtMRP2 exhibited a reduced sensitivity towards 1-chloro-2,4-dinitrobenzene, but not towards other herbicides. AtMRP2 but not AtMRP1, 11 and 12 is involved in chlorophyll degradation since ethylene-treated rosettes of atmrp2 showed reduced senescence, and AtMRP2 expression is induced during senescence. This suggests that AtMRP2 is involved in vacuolar transport of chlorophyll catabolites. Vacuolar uptake studies demonstrated that transport of typical MRP substrates was reduced in atmrp2. We conclude that within clade I, only AtMRP2 contributes significantly to overall organic anion pump activity in vivo. [ABSTRACT FROM AUTHOR]
Published: 2008
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31 results on '"Annie Frelet-Barrand"'

1. Concentration vs. Optical Density of ESKAPEE Bacteria: A Method to Determine the Optimum Measurement Wavelength

2. Absorption/Attenuation Spectral Description of ESKAPEE Bacteria: Application to Seeder-Free Culture Monitoring, Mammalian T-Cell and Bacteria Mixture Analysis and Contamination Description

3. Absorption Spectra Description for T-Cell Concentrations Determination and Simultaneous Measurements of Species during Co-Cultures

4. Lactococcus lactis, an Attractive Cell Factory for the Expression of Functional Membrane Proteins

5. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

6. Ectopic Neo-Formed Intracellular Membranes in Escherichia coli: A Response to Membrane Protein-Induced Stress Involving Membrane Curvature and Domains

7. Oligomeric status and nucleotide binding properties of the plastid ATP/ADP transporter 1: toward a molecular understanding of the transport mechanism.

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

9. Lactococcus lactis, an alternative system for functional expression of peripheral and intrinsic Arabidopsis membrane proteins.

10. Optical Spectroscopy Methods to Monitor Cells and Bacteria Concentrations and to Detect Contamination During Cell Culture: Application to the Fabrication of ATMPs.

11. Optical Spectroscopy for the Quality Control of ATMP Fabrication: A New Method to Monitor Cell Expansion and to Detect Contaminations.

12. Online Monitoring of T-Cell Growth: Assessment of the Concentration and Quality, and Real-Time Detection of Contamination

13. Optical Spectroscopy Methods to Monitor Cells and Bacteria Concentrations and to Detect Contamination during Cell Culture: Application to the Fabrication of ATMPs

14. Optical spectroscopy for the quality control of ATMP fabrication: a new method to monitor cell expansion and to detect contamination

15. Ectopic Neo-Formed Intracellular Membranes in Escherichia coli: A Response to Membrane Protein-Induced Stress Involving Membrane Curvature and Domains

16. The NOS-like protein from the microalgae Ostreococcus tauri is a genuine and ultrafast NO-producing enzyme

17. Membrane Protein Production in Lactococcus lactis for Functional Studies

18. Lactococcus lactis is an Efficient Expression System for Mammalian Membrane Proteins Involved in Liver Detoxification, CYP3A4, and MGST1

19. The Arabidopsis ATP-binding Cassette Protein AtMRP5/AtABCC5 Is a High Affinity Inositol Hexakisphosphate Transporter Involved in Guard Cell Signaling and Phytate Storage

20. Membrane Protein Production in Lactococcus lactis for Functional Studies

21. Functional Expression of Plant Membrane Proteins in Lactococcus lactis

22. Functional expression of plant membrane proteins in Lactococcus lactis

23. HMA1 and PAA1, two chloroplast envelope PIB-ATPases, play distinct roles in chloroplast copper homeostasis

24. Lactococcus lactis: Recent Developments in Functional Expression of Membrane Proteins

25. Expression of a chloroplast ATP/ADP transporter in E. coli membranes: behind the Mistic strategy

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

27. High-chloride concentrations abolish the binding of adenine nucleotides in the mitochondrial ADP/ATP carrier family

28. Membrane Protein Expression in Lactococcus lactis

29. Lactococcus lactis, an alternative system for functional expression of peripheral and intrinsic Arabidopsis membrane proteins

30. Comparative mutant analysis of Arabidopsis ABCC-type ABC transporters: AtMRP2 contributes to detoxification, vacuolar organic anion transport and chlorophyll degradation

31. Comparative Mutant Analysis of Arabidopsis ABCC-Type ABC Transporters: AtMRP2 Contributes to Detoxification, Vacuolar Organic Anion Transport and Chlorophyll Degradation.

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31 results on '"Annie Frelet-Barrand"'

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