|Production of Recombinant Proteins and Antibodies|
|HEAD||Dr. BÉGUIN Pierre / firstname.lastname@example.org|
|MEMBERS||Dr. BELLALOU Jacques / Dr. BONDET Vincent / Dr. CRUBLET Élodie / Ms. DARTEVELLE Sylvie / Ms. ESNARD Muriel / Ms GIRARD-BLANC Christine / Dr LAFAYE Pierre / Ms. LEVI-ACOBAS Fabienne / Mr. FRACHON Emmanuel / Ms HULAK Manon / Ms. LAURET Marie-Reine, Ms. MARCHAND Françoise / Dr. NATO Faridabano / Ms. NOWAKOWSKI Mireille / Mr. PÊTRES Stéphane (on leave). Ms RAZAFIMAHATRATRA Sandrine
The Platform is in charge of producing and purifying recombinant proteins and monoclonal antibodies. It comprises three modules :
1. Production of recombinant proteins in microbial systems.
Production of recombinant proteins may be performed in cultures of recombinant bacteria, yeast, or Leishmania tarentolae. Available bioreactors include shake flasks, classical 2- and 15-liter fermentors and micro-fermentor batteries whose prototype was designed and developed in-house for performing parallel cultures under defined conditions. The device features an optical system enabling on-line monitoring of the optical density of the cultures. A license has been granted to the company Fogale nanotech, who manufactures a commercial version of the battery (Figure 1).
In addition to performing to cultures under standardized conditions, a major share of the activity of the module consists in optimizing the production of "difficult" proteins that are synthesized in low amounts or in insoluble form. On demand, the Platform will perform genetic constructs for expressing the target gene in various host-vector systems.
2. Production of recombinant proteins in insect cells.
The Platform also offers the opportunity to produce recombinant proteins in baculovirus-infected cells of Spodoptera frugiperda or in stable transformed lines of Drosophila melanogaster Schneider cells. For the baculovirus system, the module performs the construction of the recombinant virus starting from a transfer vector and the production of high-titer stocks. The Platform is equipped with 20- and 50-L disposable bioreactors (Wave system) for performing large-scale cultures
3. Production of monoclonal antibodies
The module produces mouse monoclonal antibodies against antigens provided by the users. Antibodies are characterized with respect to their Ig subtype and their dissociation constant. Epitope mapping can be performed if the adequate antigens are provided.
Currently, the module manages about a dozen projects at various stages of completion. Beside production of monoclonal antibodies for research, several projects concern the development of diagnostic tests using dipsticks based on immunochromatography for the detection of pathogen-specific antigens in biological fluids. These include diagnoses for bloody diarrheas, chikungunya and leptospirosis.
Single chain camelid antibodies penetrate cells and cross the blood brain barrier
Since last year, the activity of the Platform includes basic studies on the properties of camelid single chain antibodies (VHH). These molecules offer very interesting possibilities owing to their small size and the ease with which they can be produced in recombinant E. coli. In addition, we have evidence that they can cross the blood-brain barrier : intracarotidal injection of VHH antibodies directed against glial fibrillary acidic protein enabled in vivoimmunochemical labeling of mouse brain astrocytes.
Keywords: bioreactors, recombinant protein, monoclonal antibodies, dipstick diagnosis, baculovirus, insect cells
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Vigan-Womas I, Guillotte M, Le Scanf C, Igonet S, Petres S, Juillerat A, Badaut C, Nato F, Schneider A, Lavergne A, Contamin H, Tall A, Baril L, Bentley GA, Mercereau-Puijalon O (2008). An in vivo and in vitro model of Plasmodium falciparum rosetting and autoagglutination mediated by varO, a group A var gene encoding a frequent serotype. Infect. Immun. 76, 5565-5580
Nato F, Phalipon A, Nguyen TL, Diep TT, Sansonetti P, Germani Y (2009). Dipstick for rapid diagnosis of Shigella flexneri 2a in stool. PLoS One 2, e361.
Rajerison M, Dartevelle S, Ralafiarisoa LA, Bitam I, Dinh TN, Andrianaivoarimanana V, Nato F, Rahalison L. 2009. Development and evaluation of two simple, rapid immunochromatographic tests for the detection of Yersinia pestis antibodies in humans and reservoirs. PLoS Negl Trop Dis. 3, e421.
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Activity Reports 2009 - Institut Pasteur
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