Luo D, Mah N, Wishart D, Zhang Y, Jacobs F, Martin L
Research and Development Division, Biomira Inc., Edmonton, Alberta, Canada.
J Biochem. 1996 Aug;120(2):229-32. doi: 10.1093/oxfordjournals.jbchem.a021402.
We fused various polypeptide extensions to the C-termini of single chain Fv (scFv) and disulfide-stabilized Fv (dsFv) fragments to facilitate detection of bi-functional proteins or to add biological effector domains, which included the human metallothionein (HMT) motif and biotin mimetic sequence. These bi-functional proteins were expressed and secreted in a recombinant Pichia pastoris system and showed specific anti-idiotype binding activity, as determined by competitive radioimmunoassaying. However, the fusion protein constructed with dsFv- HMT, but not scFv-HMT, had lost this binding activity. The interruption of the structural conformation as a result in dsFv-HMT may be explained by the interactions between the cysteines engineered in dsFv domains and the cysteines in the HMT region.
我们将各种多肽延伸片段融合到单链Fv（scFv）和二硫键稳定化Fv（dsFv）片段的C末端，以促进双功能蛋白的检测或添加生物效应结构域，其中包括人金属硫蛋白（HMT）基序和生物素模拟序列。这些双功能蛋白在重组毕赤酵母系统中表达并分泌，通过竞争性放射免疫测定法测定，显示出特异性抗独特型结合活性。然而，用dsFv-HMT构建的融合蛋白，而非scFv-HMT，失去了这种结合活性。dsFv-HMT中结构构象的中断可能是由于dsFv结构域中工程化的半胱氨酸与HMT区域中的半胱氨酸之间的相互作用所致。
