|Production of monoclonal antibodies and recombinant proteins|
|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 the programme dealing with the structural genomics of pathogenic Mycobacteria 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 continues to produce monoclonal antibodies and characterize their properties (binding constant, isotype, epitope mapping etc.).
During the year 2003, we developed immunochromatographic strips for the rapid diagnosis of plague and cholera. This work has been reported in three publications. Similar tests are being developed for the diagnosis of meningitis.
A monoclonal antibody specific for the recombinant protein MSP-1 P19 derived from Plasmodium falciparum was obtained. This antibody was co-crystallized with the P19 protein in the unit of Structural Immunology. This work has been reported in one publication.
We are currently involved in a Transversal Research Project with the aim to produce monoclonal antibodies neutralizing the A toxin of botulism. We also develop monoclonal antibodies against peptides derived from the PrPc protein of sheep scrapie.
Five new projects have been selected by the steering committee, who met in September 2003. Monoclonal antibodies will be raised against PfMSP4 and PfMSP5, two proteins of P. falciparum which are vaccine candidates against the blood stage of malaria, against a branched peptide derived from SUMOylated yeast proteins, against the peptide antagonist of NEP-enkephalinase in order to de velop a quantitative immunodetection assay. Further antibodies will be developed against Shigella dysenteriae 1 lipopoolysaccharide, in order to develop a rapid diagnostic test and against antigens exposed on the cell surface of erythrocytes infected by a virulent, rosette-forming clone of P. falciparum.
Production of recombinant proteins
The module Production of Recombinant Proteins performs the production of bacterial cultures in 1- to 300-liter fermentors and the preparation of crude extracts from the corresponding cell pellets. On demand, it provides expertise for optimizing the productionof recombinant proteins. It participates in the programme dealing with the structural genomics of Mycobacdterium tuberculosis and Mycobacteriumleprae.
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 functional studies or for the determination of their 3-D structure by X-ray crystallography or by NMR. 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 fermentors if larger volumes are needed.
In order to analyze rapidly how much recombinant protein is produced as a function of growth parameters, we are developing a simple and reliable technology for analytical purification of samples in parallel. On one hand, mechanical disruption ( sonication) is replaced by chemical lysis, which must be efficient, yet preserve the activity and the soluble or insoluble character of the proteins. On the other hand, we are testing various commercial systems based on metal affinity chromatography to purify and assay in parallel the recombinant protein present in each sample.
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). Thus, we purified the wild type and a mutant form of the chemokiine RANTES, which required the solubilization form inclusion bodies, the in vitro renaturation of the polypeptide along with the reoxidation of disulfide bridges and the removal of the amino-teminal methionine. The authentic character of the preparations could be verified by mass spectrometry and by their biological activity.
Furthermore, the module also takes care of the production of 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
Photo : Battery of parallel fermentors 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 2003 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)||MAIMONI-GONCALVES Viviane (firstname.lastname@example.org)||BELLALOU Jacques, (email@example.com)
BONDET Vincent (firstname.lastname@example.org)
DARTEVELLE Sylvie (email@example.com)
FRACHON Emmanuel (firstname.lastname@example.org)
IDJA Andrée (email@example.com)
MEIER Alain (firstname.lastname@example.org)
NATO Farida (email@example.com)
PETRES Stéphane (firstname.lastname@example.org)