Characterization of a progesterone-binding, three-domain antibody fragment (VH/K) expressed in <em>Escherichia coli</em>.

The heavy chain variable region (V_H) and the κ 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_H1 elbow region. The Cκ 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 κ 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/K polypeptide is assembled in the periplasm as a disulphide-bridged dimer. The V_H/K construct provides a novel route to expression of antibody combining sites in E. coli for antibody engineering. [ABSTRACT FROM AUTHOR]