Department of Biochemistry, University of Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland.
Paul Scherrer Institute, OFLC/106, 5232, Villigen PSI, Switzerland.
Sci Rep. 2017 Sep 11;7(1):11217. doi: 10.1038/s41598-017-11472-x.
Multivalent binding proteins can gain biological activities beyond what is inherent in the individual binders, by bringing together different target molecules, restricting their conformational flexibility or changing their subcellular localization. In this study, we demonstrate a method to build up rigid multivalent and multispecific scaffolds by exploiting the modular nature of a repeat protein scaffold and avoiding flexible linkers. We use DARPins (Designed Ankyrin Repeat Proteins), synthetic binding proteins based on the Ankyrin-repeat protein scaffold, as binding units. Their ease of in vitro selection, high production yield and stability make them ideal specificity-conferring building blocks for the design of more complex constructs. C- and N-terminal DARPin capping repeats were re-designed to be joined by a shared helix in such a way that rigid connector modules are formed. This allows us to join two or more DARPins in predefined geometries without compromising their binding affinities and specificities. Nine connector modules with distinct geometries were designed; for eight of these we were able to confirm the structure by X-ray crystallography, while only one did not crystallize. The bispecific constructs were all able to bind both target proteins simultaneously.
多价结合蛋白可以通过将不同的靶分子聚集在一起，限制它们的构象灵活性或改变它们的亚细胞定位，从而获得超越单个结合物固有活性的生物学活性。在这项研究中，我们展示了一种通过利用重复蛋白支架的模块性质并避免使用柔性接头来构建刚性多价和多特异性支架的方法。我们使用 DARPin（设计的锚蛋白重复蛋白）作为结合单元，这是一种基于锚蛋白重复蛋白支架的合成结合蛋白。它们易于体外选择、高生产产量和稳定性使它们成为设计更复杂构建体的理想特异性赋予构建块。C 端和 N 端 DARPin 盖帽重复序列被重新设计为通过共享螺旋连接，从而形成刚性连接器模块。这使我们能够以预定的几何形状连接两个或更多 DARPin，而不会影响它们的结合亲和力和特异性。设计了九个具有不同几何形状的连接器模块；其中有八个我们能够通过 X 射线晶体学确认结构，而只有一个没有结晶。双特异性构建体都能够同时结合两个靶蛋白。
