|Production of monoclonal antibodies and recombinant proteins (Platform)|
|Director : BÉGUIN Pierre (firstname.lastname@example.org)|
The Platform Production of Recombinant Proteins and Antibodies produces and purifies recombinant proteins as well as monoclonal antibodies. It participates in programmes dealing with the structural genomics of pathogenic Mycobacteria and the search for new drugs and diagnoses for tuberculosis, and produces monoclonal antibodies for research, diagnosis and therapy.
The Platform n° 5 comprises three modules : a) Production of monoclonal antibodies (manager : Farida Nato, email@example.com); b) Production of recombinant proteins (manager : Jacques Bellalou, firstname.lastname@example.org): purification of proteins and production of proteins in the baculovirus system (manager : Stéphane Pêtres, email@example.com)
In collaboration with various research units of the Institute, this module produces monoclonal antibodies and characterizes their properties (binding constant, isotype, epitope mapping etc.). Such antibodies are used for research and diagnosis and for the development of new therapies.
Concerning research, several projects deal with the detection of antigens present on the surface of merozoites of Plasmodium falciparum or on the surface of parasitized red blood cells. Monoclonal antibodies raised against these antigens will serve to test in vitro their ability to inhibit infection, thus validating the choice of the corresponding antigens as vaccine candidates. Other projects deal with the development of an immunoassay for sialorphine, a peptide inhibitor of enkephalinase, which modulates nociceptive signalling, and the production of antibodies against NEMO, an important mediator of the NF-κB pathway. Furthermore, we are involved in the development of antibodies specifically recognizing β-1,3/1,6-glucan branchings within the fungal cell wall.
Concerning diagnosis, the module has specialized in the development of immunochromatographic strips, which enable the detection of pathogen-specific antigens in biological fluids. Such tests are very rapid, reliable and may be carried out in the scantiest conditions. Our current projects involve the development of tests for plague, meningitis and bloody diarrhoeas. Strips are currently undergoing field evaluation for plague, meningitis caused by Neisseria meningitidis serovars A, C, Y, and W135 and for dysenteria cause by Shigella dysenteriae and Shigella flexneri. Other tests are being developed for the diagnosis of Pneumococcus, Haemophilus influenzae type B, Entamoeba histolytica, and of enterohemorrhagic strains of E. coli.
Concerning therapy, we have an ongoing project to generate antibodies neutralizing botulism toxins. Indeed, immunotherapy is currently the only cure available against this disease.
Production of recombinant proteins
The module Production of Recombinant Proteins performs the production of bacterial cultures in 1- to 300-liter fermenters and the preparation of crude extracts from the corresponding cell pellets. On demand, it provides expertise for optimizing the production of recombinant proteins. Since this year, the module also operates an Äkta chromatograph and participates in the purification of proteins.
A common objective of these activities is to reach high yields of soluble, correctly folded recombinant proteins produced in Escherichia coli. These proteins, if needed labelled with selenomethionine, are used for the determination of their 3-D structure by X-ray crystallography or by NMR or for functional studies. In order to optimize growth parameters rapidly and ensure high-throughput production, we use a battery comprising 8 miniaturized bioreactors. A computerized system controls the growth parameters of each culture independently. Sequences of growth temperature can be programmed as a function of cell density, which is measured on line. Thus, the technology is well adapted to optimize the production of poorly soluble proteins. Using High Density medium, yields of biomass and recombinant proteins obtained in one 70-ml bioreactor are similar to those obtained in 1-liter shake flasks with conventional media. Furthermore, growth conditions established in the battery may be transposed directly to cultures in classical fermenters if larger volumes are needed.
Besides numerous short-term requests for optimization and production of recombinant proteins, the module participates in a project devoted to the structural genomics of Mycobacterium tuberculosis and Mycobacterium leprae, and in a Great Horizontal Programme on tuberculosis by producing target proteins for drug screening, vaccine production, and diagnosis. Furthermore, we use the microfermentor battery to optimize culture conditions for other microorganisms beside E. coli. Thus, some target proteins of the tuberculosis program are produced in Mycobacterium smegmatis, which affords a higher yield in soluble and native protein than E. coli. Another program deals with the development of a culture medium enabling faster growth of Legionella pneumophila, in order to speed up the diagnosis of this bacterium. Besides running parallel cultures in the microfermentor battery, the project involves HPLC analysis of the medium in order to monitor the consumption of substrate and the production of metabolites.
Production and Purification of Proteins in the Baculovirus System
This module undertakes the production of proteins encoded by recombinant viruses infecting insect cells. The baculovirus system is better adapted than E. coli for the production of animal proteins, due to more closely related patterns of folding and post-translational modifications (glycosylation). Since last year, the module also uses Drosophila cells, particularly for producing viral proteins. Furthermore, it also takes on a large fraction of the purification tasks, starting either from bacterial cell pellets or from cultures of insect cells.
Besides short-term requests, the module collaborates to several mid- and long-term projects. Concerning parasitology, we strive to develop the production of the Plsmodium vivax subtilisins PvSub1 and PvSub2, which are essential to the life cycle of the parasite and represent promising drug and vaccine targets. We are also involved in the production of domains of the Plasmodium falciparum varO adhesin. Concerning cancer therapy, we have been asked to produce and purify transglycosylases derived from tumour cells, which will be used for the in vitro synthesis of glycosylated derivatives of mucin that will be assessed as anti-tumour vaccines. Concerning virology, we are involved in the production of proteins form the dengue fever virus whose three-dimensional structure is to be determined. Finally, we participate in a project studying the mechanisms of adaptation of the inner ear to sound intensity by producing proteins involved in the process, such as myosin 1c or the protein PhretB. These will be used to produce monoclonal antibodies and to identify interacting partners by co-immunoprecipitation.
Photo : Battery of parallel fermenters for high-throughput bacterial cultures. The fermentor unit is on the left, the control unit is on the right.
Keywords: Fermentors, recombinant proteins, automated cultures , protein purification, baculovirus, monoclonal antibodies, structural genomics
|More informations on our web site|
|Publications 2005 of the unit on Pasteur's references database|
|Office staff||Researchers||Scientific trainees||Other personnel|
|ESNARD Muriel (firstname.lastname@example.org)||BÉGUIN Pierre (email@example.com)||JAUNASSE Lydie, DESS Cell Engineering, U. Henri Poincaré, Nancy 1
MAHAMANE, Ali Elhadji, Biological Engineer, CERMES, Niger
MARIMOUTIN Karine, Licence Professionnelle de Biologie Appliquée, U. Paris-Sud 11
|BELLALOU Jacques, Engineer, (firstname.lastname@example.org)
BONDET Vincent, Engineer, (email@example.com)
DARTEVELLE Sylvie, Technician, (firstname.lastname@example.org)
FOURNIE-AMAZOUZ Edith, Technician, (email@example.com)
FRACHON Emmanuel, Engineer, (firstname.lastname@example.org)
LAURET Marie-Reine, Laboratory Assistant (email@example.com)
MARCHAND Françoise, Technician, Institut Pasteur (firstname.lastname@example.org)
NATO Farida, Engineer, (email@example.com)
NOWAKOWSKI Mireille, Technician (firstname.lastname@example.org)
PETRES Stéphane, Technician, Institut Pasteur, (email@example.com)