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  Director : Béguin Pierre (beguin@pasteur.fr)


  abstract

 

The Technical Platform n° 5 comprises three modules : a) Production of monoclonal antibodies (head : Farida Nato, fnato@pasteur.fr); b) Production of recombinant proteins (head : Jacques Bellalou, bellalou@pasteur.fr): purification of proteins and production of proteins in the baculovirus system (head : Pierre Béguin, beguin@pasteur.fr)



  report

cale

Monoclonal Antibodies

The module Production of monoclonal antibodies takes on the production of mouse hybridomas for scientists of the Institute and the characterisation of the antibodies with respect to class determination, dissociation constant, and recognition of specific epitopes. Monoclonal antibodies are essential reagents in diagnosis and we developed rapid tests based on immunochromatography for the diagnosis of plague and cholera. They are also widely used in basic research to analyse structure-function relationships in various model systems. Indeed, these tools are requested by groups interested in the mode of entry of certain pathogens such as Listeria monocytogenes or Shigella flexneri into epithelial cells. We also collaborate with groups looking for vaccinating antigens and participate in cross-disciplinary research programmes. One project deals with the mapping of exposed regions in the scrapie prion protein PRPSC. Our task consists in developing monoclonal antibodies directed against synthetic peptides in order to identify epitopes that are exposed on the surface of the fibrils.

Production of Recombinant Proteins

The module Production of recombinant proteins takes on for the scientists of the Institute the production of bacterial cultures in 1- to 300-liter fermenters and the preparation of crude extracts from the corresponding cell pellets.

The module also participates in the programme dealing with the structural genomics of pathogenic Mycobacteria: Mycobacterium tuberculosis et Mycobacterium leprae. It takes on the high-throughput production of bacterial cell pellets containing proteins from these organisms produced in Escherichia coli from genes cloned in expression vectors. In order to achieve high-level production of the recombinant proteins in soluble, correctly folded form, culture parameters such as medium, temperature, duration of the induction phase need to be optimized, as well as the constructs and the host cells expressing the target genes (co-expression of chaperone genes, protease-deficient strains etc.) Once conditions are established to produce proteins that can be purified and subjected to structural analysis, the proteins are labelled with15N nitrogen or selenomethionine in order to facilitate NMR or X-ray crystallographic analysis.

In order to meet requirements for high-throughput production under standardised conditions, the module is developing a system for parallel cultivation comprising a battery of eight 60-ml bioreactors. Each reactor is fitted with an independent, programmable temperature control. The optical density of each culture is followed continuously by a scanning optic sensor. Microprobes enable the monitoring of pH and pO2. An interface software has been developed in order to program culture protocols suitable for recombinant protein production and minimise hands-on intervention by the operator. A more complete version of the system will include a robot for automatic injection and sampling, which will be controlled by time or bacterial density parameters predefined by the user.

Purification of Proteins and Production in the Baculovirus System

The module Purification of proteins and protein production in the baculovirus system takes on the purification of recombinant proteins from the bacterial cell pellets obtained by the module Production of recombinant proteins. This assignment comprises the monitoring of the yield and purity of the preparations. On this account, it contributes to the programme for structural genomics of pathogenic Mycobacteria : purified proteins are transferred to the Technical Platform n° 6 for crystallization trials and X-ray diffraction analysis. Technological developments strive to automate purification procedures using a programmable Äkta Explorer chromatography system and to devise techniques for the proteolytic removal of affinity tags.

Furthermore, the module also takes on , upon request, the production of proteins expressed from recombinant baculovirus in insect cells. This task involves the construction of the recombinant vector by co-transfection and in vivo recombination and the production of recombinant proteins from infected cultures. Stable cultures of transfected cells can also be obtained.

Photo 1 : Battery of parallel fermenters for high-throughput bacterial cultures.

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  personnel

  Office staff Researchers Scientific trainees Other personnel
  ESNARD Muriel, mesnard@pasteur.fr BÉGUIN Pierre Research scientist , beguin@pasteur.fr EHSANI Parastoo Post-doctoral fellow (Tehran), pehsani@pasteur.fr BELLALOU Jacques Engineer, bellalou@pasteur.fr

BONDET Vincent Engineer, vbondet@pasteur.fr

FRACHON Emmanuel Engineer, efrachon@pasteur.fr

MEIER Alain Engineer , ameier@pasteur.fr

NATO Farida Engineer, fnato@pasteur.fr

DARTEVELLE Sylvie Technician, sdarteve@pasteur.fr

GRÉGOIRE Josiane Technician, jgregoir@pasteur.fr

PETRES Stéphane Technician, spetres@pasteur.fr

IDJA Andrée Laboratory Assistant , aidja@pasteur.fr


Activity Reports 2002 - Institut Pasteur
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