title: Novel p16 binding peptide development for p16-overexpressing cancer cell detection using phage display.
abstract: Protein p(16INK4a) (p16) is a well-known biomarker for diagnosis of human papillomavirus (HPV) related cancers. In this work, we identify novel p16 binding peptides by using phage display selection method. A random heptamer phage display library was screened on purified recombinant p16 protein-coated plates to elute only the bound phages from p16 surfaces. Binding affinity of the bound phages was compared with each other by enzyme-linked immunosorbent assay (ELISA), fluorescence imaging technique, and bioinformatic computations. Binding specificity and binding selectivity of the best candidate phage-displayed p16 binding peptide were evaluated by peptide blocking experiment in competition with p16 monoclonal antibody and fluorescence imaging technique, respectively. Five candidate phage-displayed peptides were isolated from the phage display selection method. All candidate p16 binding phages show better binding affinity than wild-type phage in ELISA test, but only three of them can discriminate p16-overexpressing cancer cell, CaSki, from normal uterine fibroblast cell, HUF, with relative fluorescence intensities from 2.6 to 4.2-fold greater than those of wild-type phage. Bioinformatic results indicate that peptide 'Ser-His-Ser-Leu-Leu-Ser-Ser' binds to p16 molecule with the best binding score and does not interfere with the common protein functions of p16. Peptide blocking experiment shows that the phage-displayed peptide 'Ser-His-Ser-Leu-Leu-Ser-Ser' can conceal p16 from monoclonal antibody interaction. This phage clone also selectively interacts with the p16 positive cell lines, and thus, it can be applied for p16-overexpressing cell detection.
authors.affiliation: NECTEC, National Science and Technology Development Agency (NSTDA), Pathumthani, 12120, Thailand; Advanced Imaging Research Center, Faculty of Medicine, Chulalongkorn University, Bangkok, 10330, Thailand.
authors.forename: Numfon
authors.initials: N
authors.lastname: Khemthongcharoen
authors.forename: Athisake
authors.initials: A
authors.lastname: Ruangpracha
authors.forename: Pongsak
authors.initials: P
authors.lastname: Sarapukdee
authors.forename: Santi
authors.initials: S
authors.lastname: Rattanavarin
authors.forename: Romuald
authors.initials: R
authors.lastname: Jolivot
authors.forename: Ungkarn
authors.initials: U
authors.lastname: Jarujareet
authors.forename: Kitiporn
authors.initials: K
authors.lastname: Plaimas
authors.forename: Parvapan
authors.initials: P
authors.lastname: Bhattarakosol
authors.forename: Suthiluk
authors.initials: S
authors.lastname: Patumraj
authors.forename: Wibool
authors.initials: W
authors.lastname: Piyawattanametha
mesh_terms: D002460:Cell Line; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000737:chemistry*; D006801:Humans; D062105:Molecular Docking Simulation; D009369:Neoplasms / Q000175:diagnosis* / Q000378:metabolism; D019151:Peptide Library; D011485:Protein Binding
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16; D019151:Peptide Library
keywords: cervical cancer; p16; peptide-protein docking; phage display; surface alignment