Efficient refolding of a recombinant abzyme

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.

Multitarget selection of catalytic antibodies with β‐lactamase activity using phage display

“…Concerning the scFv P90C2, kinetic parameters were determined using 7 μ m of soluble and purified scFv and 0.5 μ m of fluorocillin, according to Ben Naya et al . .…”

“…Kinetic measurements were performed similar to a previous publication . Briefly, the reaction was followed in 0.5 mL quartz cuvettes at RT in PBS (pH 7.4)–0.1% BSA, using a Varian Cary Eclipse Fluorescence Spectrophotometer with excitation at 495 nm and emission at 525 nm.…”

“…Most of the measurements were performed using the scFv presented on phage surface (~5 9 10 13 phage particlesÁmL À1 ). Concerning the scFv P90C2, kinetic parameters were determined using 7 lM of soluble and purified scFv and 0.5 lM of fluorocillin, according to Ben Naya et al [64].…”