The heavy chain (Hc) and light chain (Lc) genes of the Fab fragment of a catalytic antibody 6D9 were simultaneously expressed in an Escherichia coli in vitro transcription/translation system without a reducing agent. The intermolecular disulfide bond between the Hc and Lc was found formed, suggesting a correct formation of the Fab fragment in the in vitro system. In enzyme-linked immunosorbent assay, the Fab fragment synthesized in vitro exhibited an antigen-binding activity. Addition of reduced glutathione, oxidized glutathione, protein disulfide-isomerase and molecular chaperones, GroEL and GroES, increased the solubility and the antigen-binding activity of the Fab fragment greatly. The in vitro synthesized Fab was purified by means of a hexa-histidine tag attached to the C-terminus of the Hc. Catalytic assay of the purified Fab fragment showed that the His-tagged Fab fragment synthesized in vitro had a catalytic activity comparable to that produced in vivo.
