Figure 1: Schematic representation of the yeast display screening.

(a) Schematic representation of the dual promoter yeast expression plasmid encoding the C05 Fab is shown. The arrows indicate the locations of the SfiI sites employed for cloning. (b) Saturation mutagenesis was applied to the six amino acids of C05 CDR H3 that interact closely or are buried in the receptor-binding site of influenza hemagglutinin (HA). A C05 Fab plasmid mutant library, which consists of at least 10⁸ clones, was created. The plasmid mutant library was transformed into yeast strain EBY100 to generate a yeast surface display library. Different variants of C05 Fab were displayed on yeast cells. This yeast surface display library was then subjected to selection for HA-binding affinity. We used fluorescence-activated cell sorting (FACS) to enrich yeast cells that were able to interact with PE-conjugated influenza HA. The post-selection pool was then expanded and subjected to another round of selection. For each of H1, H3, and H5 HAs, three rounds of selection was performed. Variants that were able to bind to the HA would enrich in occurrence frequency throughout the screening process. Variants with higher affinity would enrich to a higher frequency. The plasmid mutant library and each of the post-selection mutant libraries were next-generation sequenced to monitor the frequency change of each variant.