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

  • 1 April 1995
    • journal article
    • research article
    • Vol. 84  (4) , 662-668
Abstract
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 secreted into the periplasmic space of Escherichia coli (E. coli). The linker 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 enzyme-linked immunosorbent assay (ELISA). Periplasmic extracts of transformed E. coli contained material that bound progesterone and related steroids with similar specificity and affinity to DB3, and displayed the DB3 idiotype and kappa chain epitopes. Reference to the crystal structure of DB3 suggests that all the characteristics of the combining site interaction with steroids are retained in the bacterially expressed material. Western blotting demonstrated material with a molecular weight equivalent to three domains after reduction, but six domains in the unreduced state, suggesting that the V-H/kappa polypeptide is assembled in the periplasm as a disulphide-bridged dimer. The V-H/kappa construct provides a novel route to expression of antibody combining sites in E. coli for antibody engineering.