Improvement of Antibody Affinity and Using PURE Ribosome Display and Microbial Secretion System

A method has been developed for efficiently enriching and analyzing high-affinity antibody variants by combining the PURE ribosome display (ribosome display using purified cell-free protein synthesis components) with next-generation sequencing (NGS) and the Brevibacillus choshinensis secretion system, using the NZ-1 antibody, which targets the PA tag peptide (GVAMPGAEDDVV), as a model antibody. An artificial DNA encoding the single-chain fragment of binding (scFab) of the NZ-1 antibody was synthesized and actively expressed by cell-free protein synthesis system (CFPS). Region-specific saturation mutations were introduced into the scFab gene based on its structural information. The resulting scFab library was selected against the PA tag through two rounds of PURE ribosome display, followed by Illumina sequencing to identify potential scFab variants with higher affinity. The candidates were expressed as Fab fragments using the B. choshinensis secretion system. These Fab fragments were then purified from the culture supernatant using two-step column chromatography. The binding affinity of the purified Fab was evaluated using a biolayer interferometry assay, revealing a variant Fab with higher affinity than the wild-type Fab. These results demonstrate that integrating PURE ribosome display with NGS analysis and the B. choshinensis secretion expression system enables the rapid identification and analysis of high-affinity antibody variants.