Strong fluorescence was observed in cells harboring the plasmid encoding gFPS, but not in the cells harboring the control plasmid, indicating that gFPS was successfully displayed on the yeast cell surface. Furthermore, when the yeast cells displaying HER2-BP 1 in gFPS were treated with rhodamine-labeled extracellular domain of HER2 protein, red fluorescence was observed only in the cells displaying gFPS-HER2-BP 1, and not in the cells displaying gFPS alone. These results clearly demonstrate that gFPS represents a unique scaffold Histamine design for peptide display in a yeast display system and strongly suggest that this scaffold can be used in a wide range of high-throughput screening systems used to identify peptides that have high target affinity and specificity. In this report, we suggest the utilization of a novel protein scaffold, gFPS, for the presentation of structurally constrained peptides to effectively identify high affinity Alvelestat (AZD9668) target-binding peptides in a conventional high-throughput screening system. gFPSs presenting HER2-BP 1 and 2, mH1 and mH2, strongly bind to the HER2-expressing N87 cells, but show no interaction with HER2-negative HeLa cells, indicating that gFPS effectively presents target-binding peptides and can identify high affinity target-binding peptides that may be functional in vivo. Note that EGFP cannot be used as a scaffold because the EGFP mutant proteins with these peptides integrated into the site corresponding to site D were insoluble. mH3, mH4, and mH5 exhibited weak affinities toward HER2-expressing cells, probably due to the loss of structural freedom of the peptide in gFPS. Because HER2-BP 3, 4, and 5 are selected from unstructured peptide libraries, their binding to HER-2 might be less specific, and based on their structural freedom, they may assume conformations that would result in lax HER2 binding. By contrast, HER2-BP 1 is locally constrained by a disulfide bond, and HER2-BP 2 was originally designed as a cyclic-constrained peptide, indicating that these peptides show maximum target affinity owing to their constrained structures. Taken together, these results demonstrate that it is important to retain structural constraint during the screening process in order to identify high affinity target-binding peptides.