YEAST EXPRESSION

 

The Yeast expression technical platform is located on two sites, one being the Laboratory “Fonction et Architecture des Assemblages Macromoléculaires” at IBBMC at Université Paris Sud (FAAM) and the other being the “Laboratoire des Protéines Membranaires” at CEA Saclay (MPEX) offering different and possibly complementary host and expression systems.

Expression in Pichia pastoris

The protein production platform of the FAAM team is set up to purify proteins obtained by recombinant expression from the méthylotrophic yeast species P. pastoris. The proteins are usually secreted in the medium. We offer the possibility to label these proteins with selenomethionine for crystallographic purposes or with isotopic  ¹⁵N/¹³C labels for NMR studies. The platform prepares the cloning into pPIC9 ou pPICZ plasmids, selects for producer clones, prepare cellular cultures (1 to 5 liter volumes using fermentors (DASGIP (4x1L) or APPLIKON (2x5L)) either in batch or in fed-batch mode. Fermentation is followed by extraction, purification, concentration and quality control of the final protein products. Extraction from cells or membrane fractions uses French press or cell fractionation with beads. Secreted proteins are isolated from the supernatant by filtration or tangential ultrafiltration (Quixstand). We dispose of 4 chromatographic purification units FPLC-AKTA, aligning affinity chromatography, gel filtration and ion-exchange columns. Proteins are concentrated by ultracentrifugation. The purity and oligomeric states of the samples are checked by SDS-PAGE, Western blotting and SEC-MALLS.

Expression in Saccharomyces cerevisiae

The « MPEX » technical Platform, located at the “Laboratoire des Protéines Membranaires” in CEA Saclay, is dedicated to expression and overexpression of soluble and transmembrane proteins. The baker’s yeast,Saccharomyces cerevisiaeis used as the main host. The bacteria E. coli could be used sporadically in some cases. The platform is designed for expression of 0.5 to 20 litres cultures. A Infors HT Multifors 2 system is available for development of protocol. This system is especially designed for multiple, parallel bioprocesses in small volumes making possible comparison of different conditions of culture or comparison of different strains. Working volumes range from 320-1000 mL. In parallel, a Infors HT Techfors-S system is available too, for already developed protocols. This system is designed for large scale production with its 20 litres containing vessel. The two systems are regulated in temperature from 6 to 37°C. pH, pO2 and temperature could be followed continually. Sampling is possible for growth monitoring and extra analysis (glucose or ethanol assay for example).

 

References (selected from the last 5 years)

Montigny, Cédric, Dong Liang Huang, Veronica Beswick, Thomas Barbot, Christine Jaxel, Marc le Maire, Ji-Shen Zheng, and Nadège Jamin. 2021. “Sarcolipin Alters SERCA1a Interdomain Communication by Impairing Binding of Both Calcium and ATP.” Scientific Reports 11 (1): 1641. https://doi.org/10.1038/s41598-021-81061-6.

Timcenko, Milena, Thibaud Dieudonné, Cédric Montigny, Thomas Boesen, Joseph A. Lyons, Guillaume Lenoir, and Poul Nissen. 2021. “Structural Basis of Substrate-Independent Phosphorylation in a P4-ATPase Lipid Flippase.” Journal of Molecular Biology, May, 167062. https://doi.org/10.1016/j.jmb.2021.167062.

Lamy, Anaïs, Ewerton Macarini-Bruzaferro, Thibaud Dieudonné, Alex Perálvarez-Marín, Guillaume Lenoir, Cédric Montigny, Marc le Maire, and José Luis Vázquez-Ibar. 2021. “ATP2, The Essential P4-ATPase of Malaria Parasites, Catalyzes Lipid-Stimulated ATP Hydrolysis in Complex with a Cdc50 β-Subunit.” Emerging Microbes & Infections 10 (1): 132–47. https://doi.org/10.1080/22221751.2020.1870413.

Geurts, Maxwell M. G., Johannes D. Clausen, Bertrand Arnou, Cédric Montigny, Guillaume Lenoir, Robin A. Corey, Christine Jaxel, et al. 2020. “The SERCA Residue Glu340 Mediates Interdomain Communication That Guides Ca2+ Transport.” Proceedings of the National Academy of Sciences 117 (49): 31114–22. https://doi.org/10.1073/pnas.2014896117.

Timcenko, Milena, Joseph A. Lyons, Dovile Januliene, Jakob J. Ulstrup, Thibaud Dieudonné, Cédric Montigny, Miriam-Rose Ash, et al. 2019. “Structure and Autoregulation of a P4-ATPase Lipid Flippase.” Nature 571 (June): 366–370. https://doi.org/10.1038/s41586-019-1344-7.

Montigny, Cédric, Thibaud Dieudonné, Stéphane Orlowski, José Luis Vázquez-Ibar, Carole Gauron, Dominique Georgin, Sten Lund, et al. 2017. “Slow Phospholipid Exchange between a Detergent-Solubilized Membrane Protein and Lipid-Detergent Mixed Micelles: Brominated Phospholipids as Tools to Follow Its Kinetics.” Edited by Hendrik W. van Veen. PLOS ONE 12 (1): e0170481. https://doi.org/10.1371/journal.pone.0170481.

Chaptal, Vincent, Frédéric Delolme, Arnaud Kilburg, Sandrine Magnard, Cédric Montigny, Martin Picard, Charlène Prier, et al. 2017. “Quantification of Detergents Complexed with Membrane Proteins.” Scientific Reports 7 (February): 41751. https://doi.org/10.1038/srep41751.

Azouaoui, Hassina, Cédric Montigny, Thibaud Dieudonné, Philippe Champeil, Aurore Jacquot, José Luis Vázquez-Ibar, Pierre Le Maréchal, et al. 2017. “A High and Phosphatidylinositol-4-Phosphate (PI4P)-Dependent ATPase Activity for the Drs2p/Cdc50p Flippase after Removal of Its N- and C-Terminal Extensions.” Journal of Biological Chemistry 292 (19): jbc.M116.751487. https://doi.org/10.1074/jbc.M116.751487.

Muzzopappa, Fernando, Adjélé Wilson, Vinosa Yogarajah, Sandrine Cot, François Perreau, Cédric Montigny, Céline Bourcier de Carbon, and Diana Kirilovsky. 2017. “The Paralogs to the C-Terminal Domain of the Cyanobacterial OCP Are Carotenoid Donors to HCPs.” Plant Physiology 175 (3): 1283–1303. https://doi.org/10.1104/pp.17.01040.

Azouaoui, Hassina, Cédric Montigny, Aurore Jacquot, Raphaëlle Barry, Philippe Champeil, and Guillaume Lenoir. 2016. “Coordinated Overexpression in Yeast of a P4-ATPase and Its Associated Cdc50 Subunit: The Case of the Drs2p/Cdc50p Lipid Flippase Complex.” Methods in Molecular Biology (Clifton, N.J.) 1377: 37–55. https://doi.org/10.1007/978-1-4939-3179-8_6.