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  Director : Michel GOLDBERG (goldberg@pasteur.fr)



This Unit combines physical-chemistry, biochemistry, genetics, protein engineering and molecular modeling approaches to deal with problems related to the structure of proteins and their integration in several cellular functions such as the acquisition of their functional structure in vitro and in vivo, energetic aspects of their interactions, the atomic origin of their stability and function, the use of modified proteins carrying "grafted" artificial peptides for vaccination, and the use of modified proteins in a variety of biotechnological applications.



I- Protein folding in vitro (Michel Goldberg)

These studies aim at understanding, at a fundamental level, the mechanisms that enable a protein to acquire, in a few seconds at most, the complex three-dimensional structure, named "native", that endows it with its biological properties. The knowledge thus acquired is used to improve protein folding, in particular in industrial processes related to biotechnologies.

a- Studies on early folding intermediates (Alain Chaffotte, Valérie Guez, Michel Goldberg and Nicole Jarrett)

We continued our studies on the coupling between the formation of long range interactions (disulfide bonds) and of local structures (the alpha-helices and beta-strands of the secondary structure) during the early folding steps of a model protein, hen lysozyme. Starting from the observation that the lysozyme secondary structure forms very rapidly when the 4 disulfide bonds are preestablished, while it does not form in their absence, we tried to identify those of the disulfide bonds that are essential for the very rapid formation of the secondary structure. For that purpose, we had constructed four double mutants in which the two cysteins engaged in an individual disulfide bonds were replaced by alanines.

The effect of the suppression of each S-S bond individually has now been characterized by a variety of spectroscopic, hydrodynamic and fonctional criteria, which confirmed the close structural similarity of the natural, fully oxidized enzyme (with 4 S-S bonds) and the modified enzymes (with 3 S-S bonds). The various phases of their refolding have been monitored using a rapid mixing device that triggers refolding in less than 4 milliseconds. This year, we completed the study of of the mutant in which the disulfide between residues 64 and 80 was removed. We thus could demonstrate that suppression of this disulfide bond does not prevent the very rapid formation (in less than 4 milliseconds) of the secondary structure. The same result had been obtained for the three other disulfides. Contrarily to the disulfide between cysteines 30 and 115, or to that between cysteines 6 and 127, but as already found for disulfide 76-94, suppression of the 64-80 disulfide did not prevent the the appearance of a partially folded intermediate that slows down the folding of natural lysozyme. However, as opposed to disulfide 76-94 which destabilizes this intermediate, disulfide 64-80 stabilizes it. The presence of this disulfide (64-80) thus appears to be responsible for the rate limiting step in the folding of the natural protein.These observations build up a set of solid experimental evidence supporting the "energy landscape" model model recently proposed by theoreticians to account for the rapidity of protein folding.

To gain a better understanding of of the coupling between disulfide bonds and regain of the native tridimensional structure of the polypeptide during lysozyme folding and oxidation, we compared the kinetics of formation of disulfide bonds with the kinetics of regain of antigenic motives that can be recognized by specific monoclonal antibodies only if the protein is (at least locally) correctly folded (see figure 1)

Two antigenic motifs, present on opposite faces of the folded protein, and depending on the proper folding of two topologically distinct regions of the polypeptide chain, have been studied. The kinetics of appearance of their reactivity to two monoclonal antibodies have been observed by means of an ELISA based, pulsed immunolabeling method developped in our laboratory. These kinetics are similar to those of appearance of species with 2 SS bonds for one of the motives and of species with 3 SS bonds for the the second.

b- Production and study of a recombinant protein, candidate vaccine against malaria (Alain Chaffotte and Anne-Gaëlle Planson)

The C-terminal fragment F19 of MSP1 (Merozoïte Surface Protein) from Plasmodium falciparum, an anti- malaria vaccine candidate, has been produced as a fusion protein with MBP (Maltose binding protein) in the periplasmic space of E. coli. Insertion at the fusion site of a sequence specific for Factor Xa proteolysis allowed isolating F19. The UV-absorption characteristics of the isolated F19 fragment are compatible with the presence of several disulfide bonds. It is not aggregated as covalent disulfide bonded oligomers. The preliminary characterization showed the E. coli-produced F19 has common structural features with the homologous F19 previously produced from insect cells. In particular, both fragments react as well against monoclonal antibodies. The comparison of their TOCSY and NOESY NMR spectra showed that both fragments have identical resonances in the spectral region characteristic of b-structure. These data suggest that F19 produced in E. coli possesses the topological organization (2 EGF domains) established by X-ray crystallography for the homologous fragment produced from insect cell. To further compare both fragments, the comparison between their immunochemical reactivity towards several monoclonal antibodies is under study.

c- Kinetics of association-dissociation and folding of the oligomeric R67 DHFR (Annick Méjean and Christophe Bodenreider)

Our studies on the kinetic aspects of the dimer-tetramer equilibrium within R67 DHFR allowed us to propose an original model taking into account the influence of the pH on this interaction. Association of two dimers is the last step of R67 DHFR folding. In order to decipher the molecular events responsible for initiating R67 DHFR folding, we aim at finding out whether association of two disordered monomers leads to the polypeptide chain folding, or folding of the monomers is required for the association. For that purpose, we performed refolding kinetics at pH5, where R67 DHFR is a stable dimer. Monitoring the folding kinetics by means of spectroscopic methods (near UV circular dichroism, tryptophan fluorescence), three phases were observed: One very fast is completed in less than 4 milliseconds; a second, fast phase has a half time of 0,5 second; the third, very slow phase has a half-time of 30 seconds. The latter phase was thought to correspond to proline cis-trans isomerization. This hypothesis was confirmed by double-jump experiments and by the specific acceleration of this phase by peptidyl-prolyl isomerase. The refolding kinetics performed at several protein concentrations as well as kinetics followed by fluorescence energy transfer (between two labeled monomers) led to the conclusion that the association step corresponds to the second fast phase. A collaboration with Dr Kellersohn (Université d'Orsay) and the use of software allowing to fit sequential first order and second order reactions, led to the precise characterization of the whole process. A fraction of the secondary structure is acquired very rapidly within the monomer (k>500 s-1). A tertiary conformational rearrangement then occurs (k=30 s-1) and is followed by the association reaction (k=106 M-1.s-1). The latter is likely accompanied by the completion of the secondary structure. Furthermore, it was demonstrated that 15 % of the molecules follow a slow folding pathway (k=0,04 s-1) due to rate limiting proline isomerizations.


II- Control of the folding of bacterial envelope proteins (Jean-Michel Betton, Nathalie Sassoon, Jean-Philippe Arié, Sabine Hunke, Mireille Hervé, Nathalie Berger and Denis Phichith)

To understand how bacterial cells recognize, signal and respond to the presence of misfolded envelope proteins, we use the overexpression of a mutant of the periplasmic maltose-binding protein, named MalE31, displaying a defective folding pathway and leading to the formation of inclusion bodies. We are currently studying the effect of temperature on the bacterial growth. Indeed, when overproduced at 30°C, MalE31 did not interfere with the bacterial physiology, but at 37°C the aggregation of MalE31 becomes toxic and causes lethality. Contrary to the expectation, heat-shock conditions (growth at 42°C) rescue this lethal phenotype by increasing the degradation of MalE31. Based on these observations, we have started two complementary approaches to characterize that specific extracytoplasmic stress response: (i) a genome-wide expression analysis using DNA macroarrays (a ready-made nylon membrane of PCR-amplified ORFs from the E.coli genome). We are comparing two pools of mRNA prepared from a strain overproducing MalEw-t or MalE31, at 30°C and 37°C. (ii) a search of multicopy suppressors of the toxic phenotype linked to the formation of inclusion bodies from MalE31, at 37°C. We just finished to construct a library of E. coli DNA fragments cloned in an expression vector. Next, we shall select or screen clones which restore growth of the MalE31 overproducing strain at 37°C.

We also study the structure-function relationships of two heat-shock periplasmic proteins; FkpA and DegP that are important players in the quality control of envelope proteins. We demonstrated that the chaperone activity of FkpA, which is independent of its PPIase activity, suppresses the formation of inclusion bodies from misfolded MalE31. For the hexameric DegP protease, we hypothesized that the PDZ domains, which form the C-terminus part of this oligomeric protein, could play a critical role in substrate recognition or by mediating self-assembly of the protease. To test this hypothesis, we constructed several DegP mutants lacking one and both PDZ domains, and assessed their oligomeric structure and proteolytic activity. Using this analysis, we have defined the minimal domain in DegP required to complement a degP- strain for growth at high temperature. Deletion of the PDZ2 domain results in a functional dimeric protein. Thus, this C-terminal domain would participate in a trimeric interface. Further studies are necessary to determine how that minimal dimeric protease could function.

Finally, we participate to structural genomics in Institut Pasteur which focus on proteins from Mycobacterium tuberculosis. By using a high-throughput cloning strategy, based on the site-specific recombination of lambda phage (Gateway), we successfully cloned the first 50 targets into a bacterial expression vector. In parallel to this structural genomics effort, we are interested by alternative expression systems to produced recombinant proteins. The Rapid Translation System (or RTS from Roche) designed for protein expression by a coupled transcription-translation reaction has retained our attention. The key technology is the cell-free continuous exchange of substrates and energy components via a semi-permeable membrane. Previously, we showed that MalE31 could be expressed in this system at 0.35 mg /ml and remained fully soluble and active, even at 37°C. In collaboration with the laboratory of "Structural Chemistry of Macromolecule", we assessed the selective incorporation of heavy atoms, 15N or 13C, into specific amino acid residues. Although bacterial expression remains the most economical method from producing uniformly labeled proteins, selective labeling of proteins with one or more15N/13C enriched amino acid in E. coli is not always possible due to amino acid metabolism. We compared the efficient incorporation of 15N/13C Asp and Arg into MalE-wt between bacterial and RTS production. This isotope labeling scheme, tested with only three amino acids, was representative of amino acid metabolic pathways of E. coli. In contrast to bacterial expression, no scrambling or dilution of isotope labels was observed with RTS production. With the development of new high yield E. coli lysats, we are pursuing our evaluation with nine different amino acids (Cys, Glu, Asp, Leu, Ser, Thr, His, Gly, and Arg). Indeed, about 5 mg/ml of protein can be produced in this new lysats. High efficient incorporation of selective labels in proteins produced by RTS500 provides the means to resolve and assign the side-chain resonances in NMR spectra of larger proteins.

III- Molecular modeling of proteins assembling processes and energies (Arnaud Blondel and Roland Nageotte)

We go on developping physical theories that describe biological macromolecules with the following motivations:

- propose new methods to model the energies of protein association that would be fast and reliable enough to bypass an experimental approach. This is an important issue for the analysis of various biological processes and for the design of new drugs.

- be able to identify the relevant conformations of biological macromolecular systems. This applies to the study of the mode of association between proteins or between proteins and drugs as well as for unraveling complex mechanisms such as protein folding.

To develop quantitative methods to model the energy of association between proteins, we have chosen to pursue simultaneously the experimental and the theoretical study of a model system, the R67 dihydrolate reductase (R67 DHFR). This protein confers an antibiotic resistance and is formed by the association of 4 identical subunits. Mutations were introduced by genetic construction to probe the importance of various subunit contacts. Association between variants were identified with a combinatorial test developed in the laboratory. The association mechanism was analyzed by means of kinetic and equilibrium measurements. The energy of these associations were then characterized with a precise physical-chemical methods also developed in the laboratory. Finally, in order to test the structural effect of the mutations, several variants were crystallized in the isolated or associated form and the crystallographic refinement of the atomic structure of these molecules was undertaken.

In parallel, in order to test the theoretical methods, the energies involved in those associations were calculated by computer modeling. That required the conception and the development of a method to calculate free energy differences with a significant reduction of uncertainties. This method was extended to take into account the water molecules and long range electrostatics. It was introduced in the academic version of the CHARMM program and implemented in the laboratory to run on Unix workstations and massively parallel super-computers. During the course of the calculations, it became clear that, for obtaining precise results, it was necessary to take into account the structural relaxations due to mutations. A simulation protocol was developed for that purpose. Redundant calculations showed that our method had good convergence properties (~0.4 kcal/mol per calculation). Finally, the agreement with experimental data was good (~0.4 kcal/mol average difference).

Many results of these studies were obtained in 2000. Several specific points were further developed in 2001.

- The mathematical model used to determine the affinity constants of the complexes from the experimental data was extended to take potential parasite reactions into account. The relative importance of these reactions was determined experimentally. The robustness of this model was tested on the experimental data.

- New calculations were performed to pursue the validation of the theoretical methods on other types of associations.

Thus, the research of the last few years provided the following results:

- A set of precise experimental data allowing to test predictions on protein-protein interactions: the effects of 15 types of modification within 6 types of complexes are known for a well defined association mechanism.

- The preliminary crystallographic refinement showed little structural changes in the associated form as compared with the wild type protein.

- It seems now possible to predict, by molecular modeling, the effects of mutations on protein association with a precision comparable to that of experiments.

- In the limits of the calculations that were performed, the force field and modeling methods used in the CHARMM program give a good representation of the physics of biological macromolecules and thus seem to be relevant to other modeling problems.

It is worth pointing out that a series of calculations takes currently about 2 months on a workstation, but that on super-computers of tomorrow ( > 1 Tflops), the same calculations should take 2-3 days, indicating that studies on the effect of a mutation will soon be a lot faster by computer modeling than by an experimental approach.

Based on the observation that the methods we developed were able to describe correctly interactions between biological macromolecules of known structure, we are now focussing our interest on the identification of the biologically relevant conformations for those systems when the structure is unknown. The application field of such studies covers the search for the mode of association between proteins, or between proteins and drugs, as well as the unraveling of complex mechanisms such as protein folding. The difficulty of such systems arises from the very large number of degrees of freedom of biological systems (three position parameters per distinguishable atom). We have based our approach on the conformational search algorithm that we developed during the preceding years. Enhanced molecular dynamic was used to generate new structures which were then sorted in sub-sets by grouping/combining. With this approach, we could propose a structure for a complex allowing a better understanding of the association and of the effect of mutations. This showed the power of the method.


Our research is focused on the relations between the three-dimensional structure of proteins, their conformational stability and their mechanism of action. We use the multidisciplinary approach of protein engineering, including in vitro molecular evolution.

a- Construction of biosensors from antibodies (Martial Renard, Laurent Belkadi and Hugues Bedouelle)

In collaboration with D. Altschuh (Strasbourg) we develop a general approach to transform any antibody into a reagentless optical biosensor. The principle consists in changing a residue of the antibody, located at the periphery of the antigen binding site, into a cysteine by directed mutagenesis, then in chemically coupling a fluorophore to this cysteine. We have established design rules, based on the crystal structure of the complex between antibody and antigen, to choose the coupling site. We have validated these rules for antibody D1.3, directed against lysozyme. The results have suggested more general design rules, which could be applied to antibodies of unknown structure and are under validation. Such immunosensors could be used in the form of protein chips for the dosage of antigens.

b- Recognition between a neutralising antibody and the dengue virus (Laurent Belkadi, Patrick England and Hugues Bedouelle)

In collaboration with the Laboratory of Antibody Engineering we analyze the mechanism of recognition between a monoclonal antibody and the four serotypes of the dengue virus, to improve its protective power. In a first step, its epitope was mapped in the envelope protein of the virus (in collaboration). In a second step, we identified the residues of the antibody which are involved in the recognition of the antigen, by changing them into alanine. The residues of the antibody which are involved in the recognition of the DEN2 serotype, constitute a sub-set of those for DEN1, their energetic contribution is lower, but the preponderant residues are the same. The mutations which decrease the association rate, correspond to those which change the canonical classes of conformation of the hypervariable loops. These results will help us to target residues of the antibody for increasing its affinity or widening its specificity by mutagenesis.

c- Structure of an eubacterial tyrosyl-tRNA synthetase (Valérie Guez and Hugues Bedouelle)

Tyrosyl-tRNA synthetase (TyrRS) participates in the translation of the generic code in vivo, and constitutes a potential target for antibiotics. It comprises three structural domains: an alpha/beta domain which is characteristic of the class I aminoacyl-tRNA synthetases, an intermediate alpha-helical domain, and a C-terminal domain (100 residues) which is disordered in the crystal structure and essential for the binding of tRNA-Tyr.In collaboration with the Unit of NMR of Biomolecules we determined the three-dimensional structure of the C-terminal domain of TyrRS from Bacillus stearothermophilus. This has provided the first complete structure of an eubacterial TyrRS. Its fold is similar to those of other RNA binding proteins, but new among the aminoacyl-tRNA synthetases.


V- Recombinant toxins of therapeutic and biotechnological interest (Daniel Ladant)

In our team, we are studying the structure/function relationships of a bacterial toxin, the adenylate cyclase (AC) toxin produced by Bordetella pertussis, the causative agent of whooping cough. This toxin, one of the major virulence factors of this organism, presents several striking features: it is secreted by the virulent bacteria and it is able to enter into eukaryotic cells where, upon activation by endogenous calmodulin, it catalyzes high-level synthesis of cAMP that in turn alters cellular physiology. The AC toxin is a 1706 residues-long protein. The calmodulin-activated, catalytic domain is located in the 400 amino-proximal residues . The carboxy-terminal 1306 residues are responsible for the binding of the toxin to the target cells and the translocation of the catalytic domain across the cytoplasmic membrane of these cells.

Our current research is mainly focused on two different aspects :

a- Mechanisms of entry of the AC toxin into eukaryotic cells and applications for the delivery of T-cell epitopes into antigen presenting cells (Cécile Bauche - Sakina Gmira - Gouzel Karimova and Daniel Ladant)

From a fundamental perspective, our objective is to analyze the molecular mechanisms of the invasion of target cells by the AC toxin. Indeed, this protein is endowed with the unique capability of delivering its N-terminal catalytic domain directly across the plasma membrane of eukaryotic cells. This objective has been pursued over the last years with a particular emphasis on the engineering of recombinant toxins of immunological interest, a project developed in close collaboration with the team of Claude Leclerc (Unité de Biologie des Régulations Immunitaires, Institut Pasteur). We have shown indeed, that the AC toxin can be used to target major histocompatibility complex (MHC) class I-associated T-cell epitopes within antigen-presenting cells (dendritic cells) in order to stimulate specific cytotoxic T- cell (CTL) responses. We previously demonstrated that the AC toxin uses as cellular receptor, the aMb2 integrin, which is expressed on a restricted subset of leukocytes (including dendritic cells). The selective targeting of the AC toxin to dendritic cells in vivo is certainly one of the key particularity that explains the remarkable efficiency of this toxin as a vaccine vehicle. We are currently attempting to characterize the interaction of the AC toxin with the aMb2 integrin at the molecular level and in particular to delineate the interacting sites on both partners. Besides we have shown that large polypeptide fragments can be inserted into the AC catalytic domain without interfering with the binding of the toxin to target cells and, in some instances, without preventing the translocation of the catalytic domain into the target cells. These results illustrate the remarkable tolerance of the AC toxin to insertion of exogenous polypeptides and have direct implications for the engineering of recombinant AC toxins carrying large fragments of antigenic proteins (studies in progress). Finally we have validated an alternative approach that consists in a chemical coupling (rather than genetic grafting) of the antigenic peptides to the recombinant AC toxin. One attractive feature of this design is that a single AC toxin carrier molecule can be easily and rapidly coupled to any desired synthetic peptides.

b- AC as a signal transducer in Escherichia coli : development of screening technologies for protein-protein interactions and proteolytic activities (Nathalie Dautin - Gouzel Karimova - Agnès Ullmann and Daniel Ladant)

B. pertussis AC represents an original model of a bacterial enzyme activated by an eukaryotic protein, calmodulin. Our general objective is to unravel the molecular basis of the interaction (and subsequent activation) of AC with its activator. Previous work indicated that the catalytic domain of AC is made of two complementary fragments, named T25 and T18, that are both required for enzymatic activity. Besides, as AC produces a regulatory molecule, cAMP, that is a pleiotropic regulator of gene transcription in E. coli , we begun a few years ago to exploit this enzyme as a signal transducer to elaborate new screening technologies in E. coli with general biotechnological applications. We took advantage of the modular structure of the AC catalytic domain to design sensitive genetic screening techniques to detect either protein/protein interactions or proteolytic activities.

In the first case, called "bacterial two-hybrid system", polypeptides of interest are genetically fused to the two AC subdomains, T25 and T18, and the hybrid proteins are co-expressed in an E. coli Dcya strain. Association of the two chimeric polypeptides allows functional complementation between the two AC subdomains and restores AC enzymatic activity: the synthesized cAMP, can then activate gene transcription thus confering a particular phenotype ("Cya+")to the host cell. This can be scored easily on appropriate indicator or selective media. This methodology is particularly suited to analyze the structure/fonction relationships of proteins. In 2001, our work has been focused on the improvement of the system in order to facilitate the screening of interacting partners of given "bait" proteins. We are currently performing large scale screenings to find specific partners of selected proteins from E. coli.

The genetic screen for proteolytic activities is based on the inactivation, upon specific proteolysis, of a chimeric AC modified by the insertion between the two AC subdomains ,T25 and T18, of a polypeptide, corresponding to the cleavage site of the protease of interest. When expressed in an E. coli Dcya strain with the cognate protease, the modified AC is cleaved and inactivated. The resulting change in the phenotype of the host cells ("Cya+" -> "Cya-") can be easily detected on indicator or selective media. To set up this genetic system we used as a model, the protease of the human immunodeficiency virus (HIV). We have shown in particular that this genetic test can detect HIV protease variants that are resistant to the anti-protease inhibitors used in AIDS-treatments. In 2001, we exploited this genetic assay to analyze the structural and functional consequences for the HIV protease of mutations that confer resistance to given drugs. We demonstrated that the polypeptide sequences adjacent to the protease in the polyprotein precursor are important for the dimerization and activity of certain protease mutants. We are also working on the development of diagnostic tests able to detect, in patients undergoing highly active anti-retroviral therapy (HAART), the emergence of HIV variants harboring antiprotease-resistant proteases.


VI- Contribution of physical-chemical methods from the Unit to various collaborationS (Alain Chaffotte - Michel Goldberg - Roland Nageotte)

The Unit offers to the scientific community, in particular the pasteurian one, its equipments and expertise in the physical-chemical studies of proteins in solution and of their interactions. It contributes to the conception, of experiments using analytical ultracentrifugation, fluorescence spectroscopy, circular dichroism, and stopped-flow rapid mixing, it performs and interprets the corresponding experiments for the benefit of laboratories on and off campus.


VII- Teaching and Education

The Unit is in charge of organizing the "Protein Biochemistry" laboratory course (Director: Alain Chaffotte) of the Institut Pasteur, which is associated to the Master's program of the Paris 6, Paris 7 and Orsay Universities, the Ecole Normale Supérieure, the Ecole Polytechnique, and the CEA. A large part of this course has been performed by members of this Unit. In particular, two groups of the Unit (Folding in vitro and Protein Folding in the bacterial envelope) have each organized 2 full weeks of laboratory sessions for the 2001 course.

1 Master students and 5 PhD students were under training in the Unit during the academic year 2000-2001.


Photo :

Figure 1: Antigenic motive of lysozyme recognized by monoclonal antibody D.1.3.

The atoms of lysozyme that contact the antibody (antigenic motive) are represented in colours. The petidic backbone is represented by white bars. This backbone must fold into a precise conformation to bring together the yellow atoms carried by one end of the chain and the red atoms carried by the other end, and position them precisely enough to form the structural motive recognized by the antibody.


puce Publications of the unit on Pasteur's references database


  Office staff Researchers Scientific trainees Other personnel

LENOIR Lucile (llenoir@pasteur.fr)

BEDOUELLE, Hugues, CNRS (hbedouel@pasteur.fr)

BETTON, Jean-Michel, CNRS (jmbetton@pasteur.fr)

BLONDEL, Arnaud, IP (ablondel@pasteur.fr)

CHAFFOTTE, Alain-François, IP (chaffott@pasteur.fr)

ENGLAND, Patrick, IP (england@pasteur.fr)

GOLDBERG, Michel, IP (goldberg@pasteur.fr)

GUEZ, Valérie, IP

KARIMOVA, Gouzel, IP (karimova@pasteur.fr)

LADANT, Daniel, CNRS (ladant@pasteur.fr)

MEJEAN, Annick, UP7 (amejean@pasteur.fr)

DE ALMEIDA, Paulo Cezar, Postdoct

ARIE, Jean-Philippe, PhD student

BAUCHE, Cécile, Postdoct

BODENREIDER, Christophe, PhD student

DAUTIN, Nathalie, PhD student

HERVE, Mireille, Researcher

HUNKE, Sabine, Postdoct

JARRETT, Nicole, PhD student

PHICHITH, Denis, Student

PLANSON, Anne-Gaëlle, PhD student

RENARD, Martial, PhD student

ULLMANN, Agnès, Emeritus Professor

BELKADI, Laurent, Engineer CNRS (belkadi@pasteur.fr)

NAGEOTTE, Roland, Engineer IP (nageotte@pasteur.fr)

SASSOON-CLAVIER, Nathalie, Technician IP (nsassoon@pasteur.fr)

NAVARRO-MARTINEZ, Maria, Responsable de Préparation IP

NGUYEN, Huu Huan, Agent Hospitalier IP

NINO, Marguerite, Agent de Laboratoire IP

THIBAUT, Florent, Agent d’Exploitation Qualifié, IP

TOMMASINO, Patrice, Agent de Laboratoire, IP


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