Fine mapping of the antigen–antibody interaction of scFv215, a recombinant antibody inhibiting RNA polymerase II from Drosophila melanogaster
Fine mapping of the antigen–antibody interaction of scFv215, a recombinant antibody inhibiting RNA polymerase II from Drosophila melanogaster
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scFv215 的抗原-抗体相互作用精细图谱，scFv215 是一种抑制黑腹果蝇 RNA 聚合酶 II 的重组抗体
DOI：
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发表时间：
1999
影响因子：
2.7
通讯作者：
S. Dübel
中科院分区：
文献类型：
--
作者：
Zhihong Liu;D. Song;A. Kramer;Andrew C. R. Martin;T. Dandekar;J. Schneider;E. Bautz;S. Dübel
分析了针对果蝇 RNA 聚合酶 II 最大亚基的细菌表达单链抗体 (scFv215)。通过链改组和位点特异性诱变来探测 scFv215 中抗原结合位点的结构和功能。重链或轻链的整个可变区被不相关的重链或轻链取代。两种替换均导致结合活性完全丧失，表明抗原结合位点是由两条链贡献的。通过对每个CDR分别进行位点特异性诱变来研究每个互补决定区(CDR)的功能贡献。其中两个 CDR（轻链 CDR1 和重链 CDR2）的突变显着降低了结合活性。这两个 CDR 中的每个氨基酸均被丙氨酸单独取代（丙氨酸步移）。发现两个 CDR 中的 7 个氨基酸取代使结合活性降低了 50% 以上。该数据支持 scFv215 的计算机模型，该模型适合基于表位突变分析的表位模型，表明主接触区域具有 α 螺旋结构。版权所有 © 1999 约翰·威利父子有限公司
A bacterially expressed single chain antibody (scFv215) directed against the largest subunit of drosophila RNA polymerase II was analysed. Structure and function of the antigen binding site in scFv215 were probed by chain shuffling and by site‐specific mutagenesis. The entire variable region of either the heavy or light chain was replaced by an unrelated heavy or light chain. Both replacements resulted in a total loss of binding activity suggesting that the antigen binding site is contributed by both chains. The functional contributions of each complementarity determining region (CDR) were investigated by site specific mutagenesis of each CDR separately. Mutations in two of the CDRs, CDR1 of light chain and CDR2 of heavy chain, reduced the binding activity significantly. Each of the amino acids in these two CDRs was replaced individually by alanine (alanine walking). Seven amino acid substitutions in the two CDRs were found to reduce the binding activity by more than 50%. The data support a computer model of scFv215 which fits an epitope model based on a mutational analysis of the epitope suggesting an alpha‐helical structure for the main contact area. Copyright © 1999 John Wiley & Sons, Ltd.
影响因子：
3.5
作者：
HO, SN;HUNT, HD;PEASE, LR
通讯作者：
PEASE, LR