title: A fluorescent protein scaffold for presenting structurally constrained peptides provides an effective screening system to identify high affinity target-binding peptides.
abstract: 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.
authors.affiliation: Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama City, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Kadonosono
authors.affiliation: Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama City, Japan.
authors.forename: Etsuri
authors.initials: E
authors.lastname: Yabe
authors.affiliation: Center for Biological Resources and Informatics, Tokyo Institute of Technology, Yokohama City, Japan.
authors.forename: Tadaomi
authors.initials: T
authors.lastname: Furuta
authors.affiliation: Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama City, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Yamano
authors.affiliation: Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama City, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Tsubaki
authors.affiliation: Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama City, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Sekine
authors.affiliation: Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama City, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Kuchimaru
authors.affiliation: Center for Biological Resources and Informatics, Tokyo Institute of Technology, Yokohama City, Japan.
authors.forename: Minoru
authors.initials: M
authors.lastname: Sakurai
authors.affiliation: Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama City, Japan.
authors.forename: Shinae
authors.initials: S
authors.lastname: Kizaka-Kondoh
mesh_terms: D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D061505:Cell Surface Display Techniques; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D009369:Neoplasms / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins
chemical_list: D002352:Carrier Proteins; D049452:Green Fluorescent Proteins; D010455:Peptides; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins
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