A fluorescent protein scaffold for presenting structurally constrained peptides provides an effective screening system to identify high affinity target-binding peptides

• Published in: PloS one 2014-08, Vol.9 (8), p.e103397-e103397
• Main Authors: Kadonosono, Tetsuya, Yabe, Etsuri, Furuta, Tadaomi, Yamano, Akihiro, Tsubaki, Takuya, Sekine, Takuya, Kuchimaru, Takahiro, Sakurai, Minoru, Kizaka-Kondoh, Shinae
• Format: Article
• Language: English
• Subjects: Affinity; Amino Acid Sequence; Amino acids; Binding; Biochemistry; Biology and Life Sciences; Biotechnology; Breast cancer; Cancer; Carrier Proteins - chemistry; Carrier Proteins - metabolism; Cell culture; Cell surface; Cell Surface Display Techniques; Epidermal growth factor; ErbB-2 protein; Fluorescence; Green fluorescent protein; Green Fluorescent Proteins - chemistry; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Health aspects; High-throughput screening; Humans; Informatics; Kinases; Libraries; Medical research; Models, Molecular; Molecular biology; Molecular dynamics; Mutation; Neoplasms - metabolism; Peptides; Peptides - chemistry; Peptides - genetics; Peptides - metabolism; Protein Binding; Protein Conformation; Protein Interaction Mapping; Proteins; Receptor, ErbB-2 - metabolism; Recombinant Fusion Proteins; Scaffolds; Screening; Target recognition; Yeast
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0103397

Peptides that have high affinity for target molecules on the surface of cancer cells are crucial for the development of targeted cancer therapies. However, unstructured peptides often fail to bind their target molecules with high affinity. To efficiently identify high-affinity target-binding peptides, we have constructed a fluorescent protein scaffold, designated gFPS, in which structurally constrained peptides are integrated at residues K131-L137 of superfolder green fluorescent protein. Molecular dynamics simulation supported the suitability of this site for presentation of exogenous peptides with a constrained structure. gFPS can present 4 to 12 exogenous amino acids without a loss of fluorescence. When gFPSs presenting human epidermal growth factor receptor type 2 (HER2)-targeting peptides were added to the culture medium of HER2-expressing cells, we could easily identify the peptides with high HER2-affinity and -specificity based on gFPS fluorescence. In addition, gFPS could be expressed on the yeast cell surface and applied for a high-throughput screening. These results demonstrate that gFPS has the potential to serve as a powerful tool to improve screening of structurally constrained peptides that have a high target affinity, and suggest that it could expedite the one-step identification of clinically applicable cancer cell-binding peptides.