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Exploration des systèmes d'expression de protéines recombinantes pour la caractérisation d'un anticorps catalytique

Abstract : Catalytic antibodies are investigated in order to understand their role under physio-pathological situations. But they also appear to be revolutionary tools to perform studies at the interface between chemistry, biochemistry, biology and immunology. Consequently, the knowledge of structure–function relationships is of great interest. We explored two expression systems for the production of a model catalytic antibody displaying a beta-lactamase activity. The recombinant scFv fragment was produced in the prokaryotic expression system. scFv fragments are often described as proteins being laborious to produce. An efficient method was developed to produce large quantities of refolded soluble catalytic scFv. Whole catalytic antibody was also produced by exploiting eukaryotic expression system. Mammalian cells were used because they are able to retain the original protein folding, assembly and post-translational modifications. The secondary structure of the catalytic scFv has been analyzed by circular dichroism to ensure that the refolded scFv is consistent with a native scFv fold. The functionality of the catalytic scFv and whole catalytic antibody has been validated by two approaches: (1) development of enzyme-linked immunosorbant assay (ELISA) and surface plasmon resonance (SPR) approaches for testing that the binding characteristics of an inhibitory peptide have been retained, and (2) proof of the subtle catalytic properties conservation through the development of a new sensitive catalytic assay using a fluorogenic substrate. This will lead to consider potential biotechnological and therapeutic applications of catalytic antibodies.
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Raouia Ben Naya. Exploration des systèmes d'expression de protéines recombinantes pour la caractérisation d'un anticorps catalytique. Sciences de l'ingénieur [physics]. Université de Technologie de Compiègne, 2013. Français. ⟨NNT : 2013COMP2078⟩. ⟨tel-00913012⟩

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