CHARACTERIZATION OF A PROGESTERONE-BINDING, 3-DOMAIN ANTIBODY FRAGMENT (V-H K) EXPRESSED IN ESCHERICHIA-COLI/

The heavy chain variable region (V-H) and the kappa light chain of the anti-progesterone monoclonal antibody (mAb) DB3, have been expressed as a single-chain three-domain polypeptide, designated V-H/K, and secr eted into the periplasmic space of Escherichia coli (E. coli). The lin ker sequence was derived from the V-H-C(H)1 elbow region. The C-kappa domain provides a sensitive detection tail for Western blotting and en zyme-linked immunosorbent assay (ELISA). Periplasmic extracts of trans formed E. coli contained material that bound progesterone and related steroids with similar specificity and affinity to DB3, and displayed t he DB3 idiotype and kappa chain epitopes. Reference to the crystal str ucture of DB3 suggests that all the characteristics of the combining s ite interaction with steroids are retained in the bacterially expresse d material. Western blotting demonstrated material with a molecular we ight equivalent to three domains after reduction, but six domains in t he unreduced state, suggesting that the V-H/kappa polypeptide is assem bled in the periplasm as a disulphide-bridged dimer. The V-H/kappa con struct provides a novel route to expression of antibody combining site s in E. coli for antibody engineering.