Efficient refolding of a recombinant abzyme Structural and catalytic characterizations

• Published in: Applied microbiology and biotechnology Vol. 97; no. 17; pp. 7721 - 7731
• Main Authors: Ben Naya, Raouia, Matti, Kalyankumar, Guellier, Adeline, Matagne, André, Boquet, Didier, Thomas, Daniel, Friboulet, Alain, Avalle, Bérangère, Padiolleau-Lefèvre, Séverine
• Format: Journal Article Web Resource
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01-09-2013; Springer; Springer Nature B.V; Springer Verlag
• Subjects: Abzymes; Antibodies; Antibodies, Catalytic - chemistry; Antibodies, Catalytic - genetics; Antibodies, Catalytic - metabolism; beta-lactamase; beta-Lactamases - chemistry; beta-Lactamases - genetics; beta-Lactamases - metabolism; Biocatalysis; Biocatalysts; Biochemistry, biophysics & molecular biology; Biochimie, biophysique & biologie moléculaire; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Catalytic antibodies; Cloning; Cytoplasmic expression; E coli; Enzymes; Genetic engineering; Kinetics; Lactams - metabolism; Life Sciences; Microbial Genetics and Genomics; Microbiology; Peptides; Polyclonal antibodies; Protein folding; Protein Refolding; Protein Structure, Secondary; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Refolding; Sciences du vivant; Single chain variable fragment; Single-Chain Antibodies - chemistry; Single-Chain Antibodies - genetics; Single-Chain Antibodies - metabolism; Studies; Surface Plasmon Resonance; France; Single chain variable fragment scFv; Catalytic antibodies; β-Lactamase; Abzymes; Cytoplasmic expression; Refolding
• ISSN: 0175-7598; 1432-0614; 1432-0614
• DOI: 10.1007/s00253-012-4600-4

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.