Yeast library screening

We built a yeast surface display vector (pYD) that contains a GAL1/10 promoter, an Aga2 cell wall tether, and a C-terminal c-Myc tag (Supplementary Figure S10). The GAL1/10 promoter induces expression of the scFv protein in medium that contains galactose. The Aga2 cell wall tether is required to shuttle the scFv to the yeast cell surface and tether the scFv to the extracellular space. The c-Myc tag is used during the flow sort to stain for yeast cells that express in-frame scFv protein. Saccharomyces cerevisiae cells (ATCC) were electroporated (Bio-Rad Gene Pulser II; 0.54 kV, 25 uF, resistance set to infinity) with gel-purified nested PCR product and linearized pYD vector for homologous recombination in vivo. Transformed cells were expanded and induced with galactose, to generate yeast scFv display libraries.

To confirm scFv surface expression, ∼ 2 × 10⁶ cells from the expanded scFv libraries were stained with anti-c-Myc (Thermo Fisher Scientific A21281) and an AF488-conjugated secondary antibody (Thermo Fisher Scientific A11039). To select scFv-expressing cells that bind to antigen, yeast cells were stained with biotinylated antigen (7 nM final concentration for protein antigens or 50 μl for polysaccharide antigens) and then stained with phycoerythrin (PE)-streptavidin (Thermo Fisher Scientific). Cells were then flow sorted on a BD Influx (Stanford Shared FACS Facility) for double-positive cells (AF488+/PE+). Recovered clones were then plated on SD-CAA plates with kanamycin, streptomycin, and penicillin (Teknova) and expanded. The expanded first round FACS clones were then subjected to a second (and sometimes third) round of FACS with the same antigen at the same molarity (7 nM final concentration for protein antigen) or volume (50 μl for polysaccharide antigens). Plasmid minipreps (Zymo Research) were prepared from yeast recovered from the final FACS sort. Tailed-end PCR was used to add Illumina adapters to the plasmid libraries for deep sequencing (Supplementary Figure S9).