title: Vl-linker-Vh orientation-dependent expression of single chain Fv-containing an engineered disulfide-stabilized bond in the framework regions.
abstract: Single chain Fv fragments (scFv) derived from an antibody, MAb 174H.64 (Tru-ScintRSQ kit, Biomira), were constructed in both orientations, i.e. Vh-linker-Vl and Vl-linker-Vh, but only the latter form could be expressed and secreted in the recombinant Pichia pastoris system. The secreted scFv protein showed specific anti-idiotype binding activity. Additionally, the molecular graphic modeling has been used to identify a possible site for the introduction of an interchain disulfide bond in the framework region of Fv. These Cys-modifications of the sites were done using a method of PCR-mediated mutagenesis. The engineered protein (disulfide-stabilized Fv: dsFv) was expressed and tested for its binding activity. It was found that dsFv was as active as the corresponding scFv and more stable as determined by competitive radioimmunoassay.
authors.affiliation: Research and Development Division, Biomira Inc., Edmonton, Alberta, Canada.
authors.forename: D
authors.initials: D
authors.lastname: Luo
authors.forename: N
authors.initials: N
authors.lastname: Mah
authors.forename: M
authors.initials: M
authors.lastname: Krantz
authors.forename: K
authors.initials: K
authors.lastname: Wilde
authors.forename: D
authors.initials: D
authors.lastname: Wishart
authors.forename: Y
authors.initials: Y
authors.lastname: Zhang
authors.forename: F
authors.initials: F
authors.lastname: Jacobs
authors.forename: L
authors.initials: L
authors.lastname: Martin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000235:genetics* / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D005818:Genetic Engineering; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology; D007136:Immunoglobulins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000276:immunology
chemical_list: D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D007136:Immunoglobulins