Luo D, Mah N, Krantz M, Wilde K, Wishart D, Zhang Y, Jacobs F, Martin L
Research and Development Division, Biomira Inc., Edmonton, Alberta, Canada.
J Biochem. 1995 Oct;118(4):825-31. doi: 10.1093/oxfordjournals.jbchem.a124986.
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.
从抗体MAb 174H.64（Tru-ScintRSQ试剂盒，Biomira公司）衍生而来的单链Fv片段（scFv），以两种方向构建，即Vh-连接子-Vl和Vl-连接子-Vh，但只有后一种形式能够在重组毕赤酵母系统中表达和分泌。分泌的scFv蛋白表现出特异性抗独特型结合活性。此外，分子图形建模已用于确定在Fv框架区引入链间二硫键的可能位点。使用PCR介导的诱变方法对这些位点进行半胱氨酸修饰。对工程化蛋白（二硫键稳定的Fv：dsFv）进行表达并测试其结合活性。通过竞争性放射免疫测定发现，dsFv与相应的scFv活性相当且更稳定。
