Institut de Biochimie et Biophysique Moléculaire et Cellulaire, Université Paris-Sud, Orsay, France ; Unité Mixte de Recherche 8619, Centre National de Recherche Scientifique, Orsay, France.
PLoS One. 2013 Aug 27;8(8):e71512. doi: 10.1371/journal.pone.0071512. eCollection 2013.
We previously designed a new family of artificial proteins named αRep based on a subgroup of thermostable helicoidal HEAT-like repeats. We have now assembled a large optimized αRep library. In this library, the side chains at each variable position are not fully randomized but instead encoded by a distribution of codons based on the natural frequency of side chains of the natural repeats family. The library construction is based on a polymerization of micro-genes and therefore results in a distribution of proteins with a variable number of repeats. We improved the library construction process using a "filtration" procedure to retain only fully coding modules that were recombined to recreate sequence diversity. The final library named Lib2.1 contains 1.7×10(9) independent clones. Here, we used phage display to select, from the previously described library or from the new library, new specific αRep proteins binding to four different non-related predefined protein targets. Specific binders were selected in each case. The results show that binders with various sizes are selected including relatively long sequences, with up to 7 repeats. ITC-measured affinities vary with Kd values ranging from micromolar to nanomolar ranges. The formation of complexes is associated with a significant thermal stabilization of the bound target protein. The crystal structures of two complexes between αRep and their cognate targets were solved and show that the new interfaces are established by the variable surfaces of the repeated modules, as well by the variable N-cap residues. These results suggest that αRep library is a new and versatile source of tight and specific binding proteins with favorable biophysical properties.
我们之前设计了一种名为αRep 的新型人工蛋白质家族，该家族基于耐热螺旋 HEAT 样重复的一个亚群。我们现在已经组装了一个大型优化的αRep 文库。在这个文库中，每个可变位置的侧链不是完全随机的，而是根据天然重复家族侧链的自然频率，通过密码子分布进行编码。文库的构建是基于微基因的聚合，因此会产生具有可变重复数目的蛋白质分布。我们使用“过滤”程序改进了文库构建过程，只保留完全编码的模块，这些模块被重新组合以重新创建序列多样性。最终命名为 Lib2.1 的文库包含 1.7×10(9)个独立的克隆。在这里，我们使用噬菌体展示技术从之前描述的文库或新文库中选择了与四个不同的非相关预定义蛋白靶标结合的新的特异性αRep 蛋白。在每种情况下都选择了特异性结合物。结果表明，选择了具有各种大小的结合物，包括相对较长的序列，最长可达 7 个重复。ITC 测量的亲和力随 Kd 值的变化而变化，范围从微摩尔到纳摩尔。复合物的形成与结合靶蛋白的显著热稳定性相关。两个αRep 与其同源靶标之间复合物的晶体结构已经解决，结果表明新的界面是由重复模块的可变表面以及可变的 N-帽残基建立的。这些结果表明，αRep 文库是一种新的、多功能的紧密和特异性结合蛋白的来源，具有良好的生物物理特性。
