The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
Vl-Linker-Vh Orientation-Dependent Expression of Single Chain Fv Containing an Engineered Disulfide-Stabilized Bond in the Framework Regions
Dong LuoNancy MahMark KrantzKristoffer WildeDavid WishartYi ZhangFred JacobsLuis Martin
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JOURNAL FREE ACCESS

1995 Volume 118 Issue 4 Pages 825-831

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Abstract

Single chain Fv fragments (scFv) derived from an antibody, MAb 174H.64 (Tru-ScintRSQTM 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.

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© The Japanese Biochemical Society
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