Isomerization of an aspartic acid residue in the complementarity-determining regions of a recombinant antibody to human IgE: Identification and effect on binding affinity
Isomerization of an aspartic acid residue in the complementarity-determining regions of a recombinant antibody to human IgE: Identification and effect on binding affinity
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DOI：
10.1021/bi951526c
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发表时间：
1996-02-13
期刊：
影响因子：
2.9
通讯作者：
Frenz, J
中科院分区：
文献类型：
--
作者：
Cacia, J;Keck, R;Frenz, J
本报告描述了位于重组单克隆抗体互补决定区（CDR）的异构化天冬氨酸残基对抗原结合的影响。该抗体与人IgE结合，其CDR内包含两个Asp-Gly序列，但发现只有一个位点不稳定发生异构化。通过使用涉及疏水相互作用色谱法（HIC）的技术促进了不稳定序列不同的抗体片段的分离和表征，该技术将乙酰基、异乙酰基和环状酰亚胺变体与位于CDR-L1中的残基分离。分离变体用于结构表征和测定其相对抗原结合亲和力。构建具有改变的残基的突变体以消除异构化的影响，并评价其结合IgE的能力。对已发表的抗体CDR晶体结构的检查表明，非反应性残基的Asp侧链的氢键结合可能是防止异构化的限制。这里概述的策略可能被证明是通用的生化和免疫化学表征的重组抗体。
This report describes the effect on antigen binding of an isomerized aspartate residue located in the complementarity-determining regions (CDRs) of a recombinant monoclonal antibody. The antibody, which binds human IgE, contains two Asp-Gly sequences within its CDRs, but only one site was found to be labile to isomerization. Isolation and characterization of antibody fragments differing in the labile sequence were facilitated by using a technique involving hydrophobic interaction chromatography (HIC) that separates aspartyl, isoaspartyl, and cyclic imide variants to the residue located in CDR-L1. The variants were isolated for structural characterization and for determination of their relative antigen binding affinities. Mutants were constructed with altered residues to obviate the effects of isomerization and were evaluated for their ability to bind to IgE. Inspection of published crystal structures of CDRs of antibodies indicated that hydrogen binding of the Asp side chain of the unreactive residue may be the constraint that prevents isomerization. The strategy outlined here may prove to be of general utility in the biochemical and immunochemical characterization of recombinant antibodies.