Application of Next-Generation Sequencing Analysis in the Directed Evolution for Creating Antibody Mimic

Abstract

Molecular evolution with a variant library is used to obtain a protein with the binding affinity for a target molecule. In the study on antibody mimics, a scaffold protein is selected from structural database, and the selected protein is functionalized by means of the molecular evolution. In a conventional molecular evolution, sub-libraries including functional variants with higher probability than initial library is generated by selection method, and functional variants are identified after the sub-libraries are monoclonalized; whereas, next-generation sequencing (NGS) is recently applied for identifying positive clones in the sub-library. In this study, we tried to monitor how functional clones behaved in the sub-libraries in the directed evolution of antibody mimics. A M13 phage displayed library, containing approximately 1.0 x 109 variants was constructed by randomizing 11 amino acids in two loops of a scaffold protein selected from Protein Data Bank. After four rounds selection, nearly 400 clones were randomly monoclnalized from third and fourth round sub-libraries and a variant with specific binding affinity (∼3µM) was identified. NGS analysis was performed to observe the distributions of variant frequencies at each round sub-library. As a result, although the distribution has little change after the elution process at first round, subsequent phage infection and bacterial amplification processes changed the distribution of the sub-library and the obtained functional variant was ranked within tenth at all the rounds. These results indicate that the enrichment of functional variants depends on the phage infection and bacterial amplification processes at the first round.