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Vl-linker-Vh orientation-dependent expression of single chain Fv-containing an engineered disulfide-stabilized bond
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.
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.