Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France.
Arcoscreen, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.
Sci Rep. 2023 Jan 24;13(1):1339. doi: 10.1038/s41598-023-27710-4.
Scaffold-based protein libraries are designed to be both diverse and rich in functional/folded proteins. However, introducing an extended diversity while preserving stability of the initial scaffold remains a challenge. Here we developed an original approach to select the ensemble of folded proteins from an initial library. The thermostable CheY protein from Thermotoga maritima was chosen as scaffold. Four loops of CheY were diversified to create a new binding surface. The subset of the library giving rise to folded proteins was first selected using a natural protein partner of the template scaffold. Then, a gene shuffling approach based on a single restriction enzyme was used to recombine DNA sequences encoding these filtrated variants. Taken together, the filtration strategy and the shuffling of the filtrated sequences were shown to enrich the library in folded and stable sequences while maintaining a large diversity in the final library (Lib-Cheytins 2.1). Binders of the Oplophorus luciferase Kaz domain were then selected by phage display from the final library, showing affinities in the μM range. One of the best variants induced a loss of 92% of luminescent activity, suggesting that this Cheytin preferentially binds to the Kaz active site.
基于支架的蛋白质文库旨在具有多样性和丰富的功能/折叠蛋白质。然而，在保持初始支架稳定性的同时引入扩展的多样性仍然是一个挑战。在这里，我们开发了一种从初始文库中选择折叠蛋白质组合的原始方法。我们选择了来自 Thermotoga maritima 的热稳定 CheY 蛋白作为支架。多样化 CheY 的四个环以创建新的结合表面。首先使用模板支架的天然蛋白质伴侣选择产生折叠蛋白质的文库亚组。然后，基于单一限制性内切酶的基因改组方法用于重组编码这些过滤变体的 DNA 序列。总之，过滤策略和过滤序列的改组被证明可以在保持最终文库（Lib-Cheytins 2.1）中高度多样性的同时，富集折叠和稳定的序列。然后，通过噬菌体展示从最终文库中选择 Oplophorus 荧光素酶 Kaz 结构域的结合物，显示出在 μM 范围内的亲和力。最佳变体之一诱导 92%的发光活性丧失，表明这种 Cheytin 优先结合 Kaz 活性位点。
