Abstract
Catalytic antibodies are currently being investigated in order to understand their role under physio-pathological situations. To this end, the knowledge of structure–function relationships is of great interest. Recombinant scFv fragments are smaller and easier to genetically manipulate than whole antibodies, making them well suited for this kind of study. Nevertheless they are often described as proteins being laborious to produce. This paper describes a highly efficient method to produce large quantities of refolded soluble catalytic scFv. For the first time, the functionality of a refolded catalytic scFv displaying a β-lactamase activity has been validated by three approaches: (1) use of circular dichroism to ensure that the refolded had secondary structure consistent with a native scFv fold, (2) development of enzyme-linked immunosorbant assay and surface plasmon resonance (SPR) approaches for testing that the binding characteristics of an inhibitory peptide have been retained, and (3) proof of the subtle catalytic properties conservation through the development of a new sensitive catalytic assay using a fluorogenic substrate.
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Acknowledgments
This work was supported by the European Union for laboratory equipment, by Conseil Regional de Picardie, grant from Agence Nationale de la Recherche (ANR-09-GENO-028). AM is supported in part by a grant from the Fonds de la Recherche Fondamentale et Collective (contract number 2.4530.09) and by the Belgian program of Interuniversity Attraction Poles initiated by the Federal Office for Scientific Technical and Cultural Affairs (PAI n° P7/xx). We thank Dr. Cyril Crosson for scientific discussions and his help in performing BIAcore experiments.
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Ben Naya, R., Matti, K., Guellier, A. et al. Efficient refolding of a recombinant abzyme. Appl Microbiol Biotechnol 97, 7721–7731 (2013). https://doi.org/10.1007/s00253-012-4600-4
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DOI: https://doi.org/10.1007/s00253-012-4600-4