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首页> 外文期刊>Journal of Molecular Biology >An antibody single-domain phage display library of a native heavy chain variable region: isolation of functional single-domain VH molecules with a unique interface.
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An antibody single-domain phage display library of a native heavy chain variable region: isolation of functional single-domain VH molecules with a unique interface.

机译：天然重链可变区的抗体单结构域噬菌体展示文库：具有独特界面的功能性单结构域VH分子的分离。

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摘要

To develop very small antibody-derived recognition units for experimental, medical, and drug design purposes, a heavy chain variable region (VH) single-domain phage-display library was designed and constructed. The scaffold that was used for library construction was a native sequence of a monoclonal antibody with a unique VH/VL interface. There was no need to modify any residues in the VL interface to avoid non-specific binding of VH domain. The library repertoire, consisting of 4x10(8)independent clones, was generated by the randomization of nine amino acid residues in complementary determining region 3. The library was screened by binding to protein antigens, and individual clones were isolated. The VH genes encoding for specific binding clones were rescued and large amounts of soluble and stable single-domain VH protein were made from insoluble inclusion bodies by in vitro refolding and purification. Biochemical and biophysical characterization of the VH protein revealed a highly specific, correctly folded, and stable monomeric molecule. Binding studies demonstrated an affinity of 20 nM. The properties of these molecules make them attractive for clinical, industrial, and research applications, as well as a step toward improvement in the design of small molecules that are based on the hypervariable loops of antibodies. Copyright 1999 Academic Press.
机译：为了开发用于实验，医学和药物设计目的的非常小的抗体衍生识别单元，设计并构建了重链可变区（VH）单域噬菌体展示文库。用于文库构建的支架是具有独特VH / VL接口的单克隆抗体的天然序列。无需修饰VL接口中的任何残基，以避免VH结构域的非特异性结合。文库库由4x10（8）个独立的克隆组成，是通过将互补决定区3中的9个氨基酸残基随机化而生成的。通过与蛋白质抗原结合来筛选文库，并分离出单个克隆。拯救了编码特异性结合克隆的VH基因，并通过体外重折叠和纯化从不溶性包涵体制备了大量可溶性和稳定的单结构域VH蛋白。 VH蛋白的生化和生物物理表征显示出高度特异性，正确折叠且稳定的单体分子。结合研究表明亲和力为20 nM。这些分子的特性使其在临床，工业和研究应用中具有吸引力，并且朝着改进基于抗体高变环的小分子设计迈出了一步。版权所有1999，学术出版社。

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