Pledger D W, Brodnicki T C, Graham B L, Tetin S, Kranz D M, Linthicum D S
Department of Veterinary Pathobiology, Texas A&M University, College Station, TX 77843, USA.
J Mol Recognit. 1999 Jul-Aug;12(4):258-66. doi: 10.1002/(SICI)1099-1352(199907/08)12:4<258::AID-JMR464>3.0.CO;2-A.
Two single-chain antibodies (scFv) that bind the superpotent sweetener ligand, NC-174, were generated from mouse monoclonal antibodies (mAb) NC6.8 (IgG, kappa) and NC10.14 (IgG, lambda). These scFv were constructed by cloning the variable region sequences of the mAb, connecting them in tandem with a 25-amino-acid polypeptide linker, and expressing them in E. coli using the pET-11a system. The recombinant proteins were purified using Ni(2+)-NTA-agarose by virtue of a hexahistidine sequence introduced to the C-terminus of the heavy chain variable region during the cloning process. The secondary structure and ligand binding properties of the two scFv, the parent mAbs and proteolytically derived Fab fragments were examined using radioligand binding, circular dichroism (CD) and fluorescence spectroscopy. The far-UV CD spectra of both scFv possessed predominantly beta character, as did those of the Fab, and the near-UV CD spectral data for scFvNC10.14, NC6.8 and NC10.14 Fab indicated that chromophore perturbation occurred upon ligand binding. The affinity constants determined for the two scFv, Fab and mAb were nearly equivalent.
从鼠单克隆抗体（mAb）NC6.8（IgG，κ）和NC10.14（IgG，λ）中制备了两种与超强甜味剂配体NC-174结合的单链抗体（scFv）。这些scFv的构建方法是：克隆mAb的可变区序列，用一段含25个氨基酸的多肽接头将它们串联连接起来，并使用pET-11a系统在大肠杆菌中进行表达。在克隆过程中，通过向重链可变区的C末端引入六组氨酸序列，利用Ni(2+)-NTA-琼脂糖对重组蛋白进行纯化。使用放射性配体结合、圆二色性（CD）和荧光光谱法，对两种scFv、亲本mAb以及经蛋白酶消化得到的Fab片段的二级结构和配体结合特性进行了检测。两种scFv的远紫外CD光谱与Fab的光谱一样，主要呈现β结构特征，scFvNC10.14、NC6.8和NC10.14 Fab的近紫外CD光谱数据表明，配体结合时发色团发生了扰动。测定得到的两种scFv、Fab和mAb的亲和常数几乎相等。
