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首页> 外文期刊>Journal of Molecular Biology >A novel type of receptor protein, based on the lipocalin scaffold, with specificity for digoxigenin.
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A novel type of receptor protein, based on the lipocalin scaffold, with specificity for digoxigenin.

机译：一种新型的受体蛋白，基于lipocalin支架，对洋地黄毒苷具有特异性。

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摘要

We demonstrate that the bilin-binding protein, a member of the lipocalin family of proteins, can be structurally reshaped in order to specifically complex digoxigenin, a steroid ligand commonly used for the non-radioactive labelling of biomolecules. 16 amino acid residues, distributed across the four loops which form the binding site of the bilin-binding protein, were subjected to targeted random mutagenesis. From the resulting library the variant DigA16 was obtained by combined use of phage display and a filter-sandwich colony screening assay, followed by in vitro affinity maturation. DigA16 possesses strong binding activity and high specificity for the digoxigenin group, with a K(D) of 30.2(+/-3.6) nM. The derivative compound digitoxigenin is bound even more tightly, with a K(D) of 2.0(+/-0.52) nM, whereas the steroid glycoside ouabain is not recognized at all. Fusion proteins between DigA16 and alkaline phosphatase were constructed and shown to retain both the digoxigenin-binding function and enzymatic activity, irrespective of whether the enzyme was fused to the N or the C terminus of the bilin-binding protein variant. Our findings suggest that the lipocalin scaffold can be generally employed for the construction of specific receptor proteins, so-called "anticalins", which provide a promising alternative to recombinant antibody fragments. Copyright 2000 Academic Press.
机译：我们证明，bilin结合蛋白，lipocalin家族蛋白的一个成员，可以进行结构重整，以特别复杂的地高辛配基，一种通常用于生物分子非放射性标记的类固醇配体。分布在形成bilin结合蛋白结合位点的四个环上的16个氨基酸残基经历了定向随机诱变。通过结合使用噬菌体展示和滤膜-夹心菌落筛选测定法，然后进行体外亲和力成熟，从得到的文库中获得变体DigA16。 DigA16对地高辛素组具有很强的结合活性和高特异性，K（D）为30.2（+/- 3.6）nM。衍生化合物洋地黄毒苷的结合更紧密，K（D）为2.0（+/- 0.52）nM，而类固醇糖苷哇巴因则根本不被识别。构建了DigA16和碱性磷酸酶之间的融合蛋白，并显示了既保持了洋地黄毒苷结合功能和酶活性，又不管该酶是否融合到胆红素结合蛋白变异体的N或C末端。我们的发现表明，脂笼蛋白支架通常可用于构建特异性受体蛋白，即所谓的“ anticalins”，其提供了重组抗体片段的有希望的替代物。版权所有2000学术出版社。

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