Characterization of a progesterone-binding, three-domain antibody fragment (VH/K) expressed in Escherichia coli.
The heavy chain variable region (VH) and the kappa light chain of the anti-progesterone monoclonal antibody (mAb) DB3, have been expressed as a single-chain three-domain polypeptide, designated VH/K, and secreted into the periplasmic space of Escherichia coli (E. coli). The linker sequence was derived from the VH-CH1 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 VH/K polypeptide is assembled in the periplasm as a disulphide-bridged dimer. The VH/K construct provides a novel route to expression of antibody combining sites in E. coli for antibody engineering.
He
M
M
Department of Immunology, Babraham Institute, Cambridge, UK.
Kang
A S
AS
Hamon
M
M
Humphreys
A S
AS
Gani
M
M
Taussig
M J
MJ
D000818:Animals; D000907:Antibodies, Bacterial / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000276:immunology; D000915:Antibody Affinity; D000918:Antibody Specificity; D001483:Base Sequence; D015153:Blotting, Western; D004926:Escherichia coli / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000378:metabolism; D007130:Immunoglobulin Idiotypes / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000378:metabolism*; D007145:Immunoglobulin kappa-Chains / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011374:Progesterone / Q000378:metabolism*
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D007130:Immunoglobulin Idiotypes; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D011374:Progesterone