Sun M, Li L, Gao Q S, Paul S
Department of Anesthesiology, University of Nebraska Medical Center, Omaha 68198.
J Biol Chem. 1994 Jan 7;269(1):734-8.
A monoclonal antibody to vasoactive intestinal polypeptide (VIP) was reduced and alkylated and its light and heavy chains were purified by denaturing gel filtration. Following renaturation, the light chain displayed sequence-specific binding of VIP. The specific VIP binding activity of several fractions spanning the light chain peak recovered from the gel filtration column was constant, the light chain was electrophoretically homogeneous, the VIP binding activity was precipitated by anti-light chain antibody but not anti-heavy chain antibody and the activity remained associated with a light chain fraction recovered by resolutive chromatography on a hydroxylapatite column. N-terminal amino acid sequencing of the light and heavy chain fractions confirmed the purity of these proteins and suggested that the VL and VH regions belonged to kappa-family II and gamma-family III, respectively. The VIP-binding affinity of the light chain was only 5-fold lower than that of the parent antibody and the light chain did not bind unrelated peptides. These observations suggest that light chains display structural characteristics necessary for high affinity antigen binding.
一种针对血管活性肠肽（VIP）的单克隆抗体经还原和烷基化处理，其轻链和重链通过变性凝胶过滤进行纯化。复性后，轻链表现出对VIP的序列特异性结合。从凝胶过滤柱回收的跨越轻链峰的几个组分的特异性VIP结合活性是恒定的，轻链在电泳上是均一的，VIP结合活性可被抗轻链抗体沉淀而不能被抗重链抗体沉淀，并且该活性仍与通过羟基磷灰石柱上的分辨色谱法回收的轻链组分相关。轻链和重链组分的N端氨基酸测序证实了这些蛋白质的纯度，并表明VL和VH区域分别属于κ家族II和γ家族III。轻链的VIP结合亲和力仅比亲本抗体低5倍，并且轻链不结合无关肽。这些观察结果表明轻链具有高亲和力抗原结合所需的结构特征。
