[2P-2] Poster 2 (2P-38ー2P-88)

[2P-79*] Application of next-generation sequencing analysis in the molecular evolution of antibody mimics

^○Tomoyuki Ito¹, Hafumi Nishi^2,3, Thuy Duong Nguyen⁴, Yutaka Saito^4,5, Tomoshi Kameda^4,5, Hikaru Nakazawa¹, Koji Tsuda^5,6, Mitsuo Umetsu^1,5 (1.Dept. of Biomol. Eng., Grad. Sch. Eng., Tohoku Univ., 2.Grad. Sch. Information Sci., Tohoku Univ., 3.Fac. Core Res., Ochanomizu Univ., 4.AIRC, AIST, 5.Adv. Intell. Pro., RIKEN, 6.Grad. Sch. Frontier Sci., The Univ. of Tokyo)

Molecular evolution with a variant library is used to obtain a molecular recognition protein. In the study on antibody mimics, a scaffold protein is functionalized by means of molecular evolution. In molecular evolution, functional variants are screened from a library, and amplified for subsequent selection rounds. Recently, next-generation sequencing (NGS) is applied to identify functional variants by analyzing enrichment of variants in the selection. Here, we tried to monitor how functional variants behaved in molecular evolution of antibody mimics. A M13 phage displayed library, containing 1.0 × 10⁹ variants was constructed by randomizing 11 amino acids in two loops of a scaffold protein. After four rounds of selection, 400 clones were randomly monoclonalized and several variants with binding affinity were identified. For a series of variant libraries in the panning rounds, we applied NGS analysis to analyze the change of variant frequencies. As a result, the frequencies of the identified variants showed continuous enrichment in a series of panning rounds; whereas the frequencies of randomly picked negative variants did not show the enrichment. These results indicate NGS analysis may be useful to classify positive and negative variants.