Expression of Fab fragment of catalytic antibody 6D9 in an Escherichia coli in vitro coupled transcription/translation system

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.

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