|Production of monoclonal antibodies and recombinant proteins (Platform)|
|Director : BÉGUIN Pierre (beguin@pasteur .fr)|
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, firstname.lastname@example.org); b) Production of recombinant proteins (manager : Jacques Bellalou, email@example.com) purification of proteins and production of proteins in the baculovirus system (manager : Stéphane Pêtres, firstname.lastname@example.org)
In collaboration with various research units of the Institute, this module produces monoclonal antibodies and characterize 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 with the mapping of surface-exposed epitopes of the pathologic form of prion protein.
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 most scanty conditions. Two projects are under way. One aims at the identification of bacterial pathogens causing meningitis, particularly Neisseria meningitidis serovars: the other seeks to diagnose pathogens responsible for bloody diarrhea: Shigella dysenteriae, enteropathogenic Escherichia coli strains, and Entamoeba histolytica.
Concerning therapy, we have an ongoing project to generate antibodies neutralizing botulism toxins. Indeed, immunotherapy is currently the only cure 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. It participates in the programme dealing with the structural genomics of Mycobacterium tuberculosis and Mycobacterium leprae. It also participates in a Great Horizontal Programme on tuberculosis by producing target proteins for drug screening, vaccine production, and diagnosis.
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.
We currently use the microfermenter battery for the majority of cultures and to optimize new culture protocols. Thus, we are currently adapting the "autoinducing medium strategy developed by F.W. Studier and colleagues to High Density medium. This approach consists in replacing isopropyl-β-thiogalactoside (IPTG) by lactose as an inducer, and in adding a limiting concentration of glucose at the beginning of the culture in order to repress expression of the target gene. Once glucose becomes exhausted, lactose penetrates the cells and is converted into allolactose, the inducer, owing to derepression of lactose permease and β-galactosidase. The technique minimizes intervention of the operator, since the inducer no longer has to be introduced at a specific optical density; furthermore, encouraging results are sometimes obtained with target proteins that are difficult to produce in soluble form.
Purification of Proteins and Production in the Baculovirus System
The module Purification of Proteins and Production in the Baculovirus System performs the purification of recombinant proteins from the bacterial cell pellets obtained by the module Production of Recombinant Proteins. The task includes monitoring of the yields and purity of the preparations. Beside requests for the purification of individual proteins, it participates in the programme dealing with the structural genomics of mycobacterial pathogens : purified proteins are delivered to the Platform Crystallogenesis and X-ray Diffraction for crystallization trials and X-ray diffraction analysis. A programmable Äkta Explorer chromatography system is available, which can be fitted with various types of columns (IMAC, ion exchange, gel filtration). On demand, we optimize the purification process and perform post-translational modifications in vitro (removal of affinity tag, controlled proteolysis, renaturation).
Furthermore, the module also produces proteins expressed by recombinant baculovirus in insect cells. This task includes the construction of the recombinant vector by co-transfection and in vivo recombination and the production of proteins for cultures of infected cells. Stable cultures of transfected cells can also be obtained. The demand for production of recombinant proteins in insect cells is growing, particularly for eucaryotic proteins, which tend to be synthesized in a more native form than in E. coli. Projects started recently include the production of Plasmodium vivax and P. falciparum proteases required for the infection of red blood cells by these parasites, the production of glycosyl transferases for the enzymatic synthesis of anti-tumor vaccines, and the synthesis of myosin forms specifically present in the inner ear.
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: Fermenters, recombinant proteins, automated cultures , protein purification, baculovirus, monoclonal antibodies, structural genomics
|More informations on our web site|
|Publications 2004 of the unit on Pasteur's references database|
|Office staff||Researchers||Scientific trainees||Other personnel|
|ESNARD Muriel (email@example.com)||BÉGUIN Pierre (firstname.lastname@example.org)||MAIMONI-GONCALVES Viviane post-doc (email@example.com)||BELLALOU Jacques, engineer (firstname.lastname@example.org)
BONDET Vincent, engineer (email@example.com)
DARTEVELLE Sylvie, technician (firstname.lastname@example.org)
FRACHON Emmanuel engineer (email@example.com)
IDJA Andrée, laboratory assistent (firstname.lastname@example.org)
MARCHAND Françoise, technician (email@example.com)
MEIER Alain, engineer (firstname.lastname@example.org)
NATO Farida, engineer (email@example.com)
PETRES Stéphane, technician (firstname.lastname@example.org)