Yamauchi Asao, Nakashima Toshihiro, Tokuriki Nobuhiko, Hosokawa Masato, Nogami Hideki, Arioka Shingo, Urabe Itaru, Yomo Tetsuya
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1, Yamada-oka, Suita Shi, Osaka, 565-0871, Japan.
Protein Eng. 2002 Jul;15(7):619-26. doi: 10.1093/protein/15.7.619.
A directed evolution with phage-displayed random polypeptides of about 140 amino acid residues was followed until the sixth generation under a selection based on affinity to a transition state analog for an esterase reaction. The experimental design deliberately limits the observation to only 10 clones per generation. The first generation consists of three soluble random polypeptides and seven arbitrarily chosen clones from a previously constructed library. The clone showing the highest affinity in a generation was selected and subjected to random mutagenesis to generate variants for the next generation. Even within only 10 arbitrarily chosen polypeptides in each of the generations, there are enough variants in accord to capacity of binding affinity. In addition, the binding capacity of the selected polypeptides showed a gradual continuous increase over the generation. Furthermore, the purified selected random polypeptides exhibited a gradual but significant increase in esterase activity. The ease of the functional development within a small sequence variety implies that enzyme evolution is prompted even within a small population of random polypeptides.
基于对酯酶反应过渡态类似物的亲和力进行筛选，对约140个氨基酸残基的噬菌体展示随机多肽进行定向进化，直至第六代。实验设计特意将每代的观察限制在仅10个克隆。第一代由三个可溶性随机多肽和七个从先前构建的文库中任意选择的克隆组成。选择在一代中显示出最高亲和力的克隆，并进行随机诱变以产生下一代的变体。即使在每代仅10个任意选择的多肽中，根据结合亲和力的能力也有足够的变体。此外，所选多肽的结合能力在各代中显示出逐渐持续增加。此外，纯化的所选随机多肽的酯酶活性也有逐渐但显著的增加。在小序列多样性内功能发展的容易程度意味着即使在少量随机多肽群体中也能促进酶的进化。
