Malby R L, Caldwell J B, Gruen L C, Harley V R, Ivancic N, Kortt A A, Lilley G G, Power B E, Webster R G, Colman P M
Biomolecular Research Institute, Parkville, Vic, Australia.
Proteins. 1993 May;16(1):57-63. doi: 10.1002/prot.340160107.
The variable heavy (VH) and variable light (VL) genes of NC10, a monoclonal antibody with specificity toward N9 neuraminidase (NA), were cloned and sequenced. A single chain Fv (scFv) fragment of NC10, consisting of VH and VL domains joined by a peptide linker, was designed, constructed and expressed in the E. coli expression vector pPOW. The N-terminal secretion signal PelB directed the synthesized protein into the periplasm where it was associated with the insoluble membrane fraction. An octapeptide (FLAG) tail was fused to the C-terminus of the single chain Fv to aid in its detection and remained intact throughout the protein purification process. NC10 scFv was purified by solubilization of the E. coli membrane fraction with guanidinium hydrochloride followed by column chromatography. The purified NC10 scFv showed binding affinity for its antigen, NA, 2-fold lower than that of the parent Fab. The complex between NA and the scFv has been crystallized by the vapor diffusion method. The crystals are tetragonal, space group P42(1)2, with unit cell dimensions a = b = 141 A, c = 218 A.
克隆并测序了对N9神经氨酸酶（NA）具有特异性的单克隆抗体NC10的可变重链（VH）和可变轻链（VL）基因。设计、构建了由通过肽接头连接的VH和VL结构域组成的NC10单链Fv（scFv）片段，并在大肠杆菌表达载体pPOW中进行表达。N端分泌信号肽PelB将合成的蛋白质引导至周质，在那里它与不溶性膜部分相关联。一个八肽（FLAG）尾巴融合到单链Fv的C端以帮助其检测，并且在整个蛋白质纯化过程中保持完整。通过用盐酸胍溶解大肠杆菌膜部分，然后进行柱色谱法来纯化NC10 scFv。纯化后的NC10 scFv对其抗原NA的结合亲和力比亲本Fab低2倍。通过气相扩散法使NA与scFv之间的复合物结晶。晶体为四方晶系，空间群P42(1)2，晶胞参数a = b = 141 Å，c = 218 Å。
