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首页> 外文期刊>Journal of Molecular Biology >Expanding the conformational diversity by random insertions to CDRH2 results in improved anti-estradiol antibodies.
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Expanding the conformational diversity by random insertions to CDRH2 results in improved anti-estradiol antibodies.

机译：通过随机插入CDRH2来扩大构象多样性，可改善抗雌二醇抗体。

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摘要

The length of the heavy chain complementarity-determining region 2 (CDRH2) was extended beyond what is found in germline genes to improve the binding properties of an anti-estradiol antibody. The previous immunochemical characterization and the molecular modeling of the high affinity (Ka=3.9x10(8)) murine anti-estradiol antibody 57-2 suggested that a part of the antigen was loosely recognized by the antibody. The CDRH2, because of its close location but scarce contacts with the hapten, was considered as a conceivable target for mutagenesis. Libraries with either two, three or four random amino acid insertions in the tip of the CDRH2 loop were constructed and displayed on the M13 filamentous phage as Fab fragments. Mutations were introduced also into the rest of the VHdomain by error-prone polymerase chain reaction to allow the surrounding structures to adapt to the extended CDRH2. After the panning of the libraries with an antigen off-rate-based selection, a number of active clones, most of which showed significantly improved affinity and specificity, were isolated, characterized and sequenced. The results indicate that the structure of the antibody can tolerate a number of different insertions in the CDRH2 region. They also suggest that the repertoire of antibody libraries can be expanded by extending the length of the CDR loops beyond that naturally provided by the given set of germline genes. This kind of mutagenesis can be generally useful for the engineering of hapten-binding antibodies. Copyright 1999 Academic Press.
机译：重链互补决定区2（CDRH2）的长度超出了种系基因中发现的长度，以改善抗雌二醇抗体的结合特性。先前的免疫化学特性和高亲和力（Ka = 3.9x10（8））鼠抗雌二醇抗体57-2的分子模型表明，该抗原的一部分被松散地识别。由于CDRH2位置靠近但与半抗原很少接触，因此它被认为是诱变的目标。构建在CDRH2环末端具有两个，三个或四个随机氨基酸插入的文库，并将其作为Fab片段展示在M13丝状噬菌体上。还通过易错聚合酶链反应将突变引入到VH结构域的其余部分，以使周围的结构适应扩展的CDRH2。用基于抗原失速率的选择淘选文库后，分离，表征和测序了许多活性克隆，其中大多数显示出显着改善的亲和力和特异性。结果表明，抗体的结构可以耐受CDRH2区域中的许多不同插入。他们还建议，可以通过将CDR环的长度扩展到超出给定种系基因组自然提供的长度来扩展抗体库的库。这种诱变通常可用于半抗原结合抗体的工程改造。版权所有1999，学术出版社。

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