title: Efficient refolding of a recombinant abzyme : structural and catalytic characterizations.
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.
authors.affiliation: Génie Enzymatique et Cellulaire (GEC), UMR 6022 CNRS, Université de Technologie de Compiègne, BP 20529, 60205, Compiègne, France.
authors.forename: Raouia
authors.initials: R
authors.lastname: Ben Naya
authors.forename: Kalyankumar
authors.initials: K
authors.lastname: Matti
authors.forename: Adeline
authors.initials: A
authors.lastname: Guellier
authors.forename: André
authors.initials: A
authors.lastname: Matagne
authors.forename: Didier
authors.initials: D
authors.lastname: Boquet
authors.forename: Daniel
authors.initials: D
authors.lastname: Thomas
authors.forename: Alain
authors.initials: A
authors.lastname: Friboulet
authors.forename: Bérangère
authors.initials: B
authors.lastname: Avalle
authors.forename: Séverine
authors.initials: S
authors.lastname: Padiolleau-Lefèvre
mesh_terms: D017151:Antibodies, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D007700:Kinetics; D007769:Lactams / Q000378:metabolism; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D017151:Antibodies, Catalytic; D007769:Lactams; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D001618:beta-Lactamases