Production of functional antigen-binding fragment of antibodies (Fab) in a cell-free protein synthesis system

Botulinum neurotoxin serotype B (boNT/B)-specific Fab was expressed in a modified extract of Escherichia coli . Toxicity of bontB was neutralized by the cell-free expressed antibody fragment. Functional Fab was obtained only when the E.coli extract was pre-treated such that its reducing activity was exhausted. Under oxidizing reaction conditions (4 mM　oxidized glutathione and 1 mM　reduced glutathione), the pre-treated extract　successfully generated active Fab molecules. Addition of the molecular chaperones (GroEL/GroES) and E.coli disulfide bond isomerase(DsbC) further increased the amount of soluble protein.In the presence of redox buffer, DsbC and chaperone, 10% of the cell-free expressed Fab (about 25 μg/ml) was recovered from nickel IMAC (immobilized metal ion affinity chromatography) column. The efficacy of the purified Fab was confirmed by the mouse protection assay.