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首页> 外文期刊>Journal of Molecular Biology >From DARPins to LoopDARPins: Novel LoopDARPin design allows the selection of low picomolar binders in a single round of ribosome display
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From DARPins to LoopDARPins: Novel LoopDARPin design allows the selection of low picomolar binders in a single round of ribosome display

机译：从DARPins到LoopDARPins：新颖的LoopDARPin设计允许在单轮核糖体展示中选择低皮摩尔结合剂

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摘要

Antibodies are the most versatile binding proteins in nature with six loops creating a flexible continuous interaction surface. However, in some molecular formats, antibodies are aggregation prone. Designed ankyrin repeat proteins (DARPins) were successfully created as alternative design solutions. Nevertheless, their concave shape, rigidity and incompletely randomized binding surface may limit the epitopes that can be targeted by this extremely stable scaffold. Combining conformational diversity and a continuous convex paratope found in many antibodies with the beneficial biophysical properties of DARPins, we created LoopDARPins, a next generation of DARPins with extended epitope binding properties. We employed X-ray structure determination of a LoopDARPin for design validation. Biophysical characterizations show that the introduction of an elongated loop through consensus design does not decrease the stability of the scaffold, consistent with molecular dynamics simulations. Ribosome-display selections against extracellular signal-regulated kinase 2 (ERK2) and four members of the BCL-2 family (BCL-2, BCL-XL, BCL-W and MCL-1) of anti-apoptotic regulators yielded LoopDARPins with affinities in the mid-picomolar to low nanomolar range against all targets. The BCL-2 family binders block the interaction with their natural interaction partner and will be valuable reagents to test the apoptotic response in functional assays. With the LoopDARPin scaffold, binders for BCL-2 with an affinity of 30 pM were isolated with only a single round of ribosome display, an enrichment that has not been described for any scaffold. Identical stringent one-round selections with conventional DARPins without loop yielded no binders. The LoopDARPin scaffold may become a highly valuable tool for biotechnological high-throughput applications.
机译：抗体是自然界中用途最广泛的结合蛋白，具有六个环，可形成灵活的连续相互作用表面。但是，在某些分子形式中，抗体易于聚集。设计的锚蛋白重复蛋白（DARPins）已成功创建为替代设计解决方案。然而，它们的凹形，刚性和不完全随机的结合表面可能会限制这种极其稳定的支架可以靶向的表位。结合在许多抗体中发现的构象多样性和连续的凸互补位以及DARPins的有益生物物理特性，我们创建了LoopDARPins，这是下一代DARPins，具有扩展的表位结合特性。我们采用LoopDARPin的X射线结构确定进行设计验证。生物物理特征表明，通过共有设计引入延长的环并不会降低支架的稳定性，这与分子动力学模拟一致。针对细胞外信号调节激酶2（ERK2）和抗凋亡调节剂的BCL-2家族的四个成员（BCL-2，BCL-XL，BCL-W和MCL-1）的核糖体展示选择产生了具有亲和力的LoopDARPins针对所有目标的中等皮摩尔至低纳摩尔范围。 BCL-2家族结合剂阻断了其与天然相互作用伴侣的相互作用，将是在功能测定中测试细胞凋亡反应的有价值的试剂。使用LoopDARPin支架，仅通过单轮核糖体展示就可以分离出亲和力为30 pM的BCL-2结合剂，但尚未针对任何支架进行过富集。与不带环的常规DARPins进行相同严格的一轮选择不会产生任何粘合剂。 LoopDARPin支架可能会成为生物技术高通量应用中极有价值的工具。

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