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FRENCH INFRASTRUCTURE FOR INTEGRATED STRUCTURAL BIOLOGY

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SOUTH PARIS

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:

Clausen, J.,et al., EMBO J,just accepted.; Berrier, C.,et al.,  J Biol Chem, 2013.; David-Bosne, S., et al.,Febs J, 2013.; Andrianaivomananjaona, T.,et al., Biochim Biophys Acta, 2011.; Arnou, B., et al., Biochem Soc Trans, 2011.; Cardi, D., et al.,  Methods Mol Biol, 2010.; Marchand, A., et al.,  J Biol Chem, 2008.

 

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