Fig. 3: Selection of yeast-encoded macrocyclic peptides towards multiple protein targets.

a General flowchart applied to identify yeast-encoded macrocyclic peptide ligands (MPs) towards five highly diverse PTs. The five yeast-encoded MP libraries (CX₇C, CX₉C, CX₃CX₉C, CX₆CX₆C, and CX₉CX₃C) were pooled together and then incubated separately with each PT through two rounds of magnetic beads (MB) separation followed by four rounds of FACS-based selection. In the flowchart of MB-based selection, the biotinylated PT immobilised on MB (PT-MB) is represented as a grey circle (PT) surrounded by a dotted ring (MB). In FACS-based selection, the soluble biotinylated PT is represented as a grey circle (PT) and the fluorescently labelled anti-HA antibody (IF) as an orange star. b Schematic representation of the iterative selection pathways applied to isolate yeast-encoded MPs against five different PTs. Two cycles of MB-based screening followed by four cycles of FACS sorts were applied. c Density plots of a representative polyclonal population of yeast cells encoding different MPs against PT5 that has been enriched from 11% to 82% through four FACS sorts (I, II, III and IV). Each dot represents two fluorescent signals of a single yeast cell. The fluorescence intensity on the y-axis is a measure of the amount of PT bound to the surface of a yeast cell (DyLight 650; ‘target binding’) whereas the fluorescence intensity on the x-axis is a measure of the number of MPs expressed on the yeast cell surface (DyLight 488; ‘macrocyclic peptide display’). d Columns graph reporting the geometric mean fluorescence (MFI) measured for the polyclonal population of yeast cells encoding different MPs against PT2 through four cycles of FACS. e Density plots of the enriched polyclonal population of yeast cells encoding different MPs against the respective PT after the fourth and last FACS cycle. f Heat map displaying the ratio between the binding and display MFI of the polyclonal population after each round of FACS. The intensity of the colour correlates with MFI value. High and low intensities are shown in dark blue and white, respectively. Data for c–f are provided as Source data.