Vl-linker-Vh orientation-dependent expression of single chain Fv-containing an engineered disulfide-stabilized bond in the framework regions.
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.
Luo
D
D
Research and Development Division, Biomira Inc., Edmonton, Alberta, Canada.
Mah
N
N
Krantz
M
M
Wilde
K
K
Wishart
D
D
Zhang
Y
Y
Jacobs
F
F
Martin
L
L
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
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D007136:Immunoglobulins