Fig. 1.

Overview of biopanning method and yeast reagent design. (a) Schematic representation of biopanning method. Wells are first coated with Matrigel, and HEK293FT cells are seeded. The cell surface is then biotinylated, and streptavidin is added. Biotinylated peptides are then added to the wells to be used as target antigens. Yeast cells displaying antibody binders and intracellularly expressing fluorescent protein are added to the wells and used as reporters. (b) Schematic representation of different reporter designs. The pBEVY backbone expresses antibody fragments (scFv) on the yeast cell surface and fluorescent protein (GFP) intracellularly. The pAP208 backbone expresses both scFv and GFP on the yeast cell surface. (c) Comparing expression levels for scFv and GFP using flow cytometry. scFv expression was measured using antibody staining of the FLAG epitope tag. Each datapoint indicates geometric mean of fluorescence from three separate experiments. Error bars indicate standard deviation of the datapoints. ^****  P ≤ 0.0001 using Tukey’s multiple comparisons test. Otherwise, P > 0.05. (d) Representative images indicating presence of streptavidin (labeled with Alexa Fluor 647) on HEK293FT cell surface (red) and yeast cells expressing GFP (cyan) binding to target peptide.