Novel p16 binding peptide development for p16‐overexpressing cancer cell detection using phage display |
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| Authors: | Numfon Khemthongcharoen Athisake Ruangpracha Pongsak Sarapukdee Santi Rattanavarin Romuald Jolivot Ungkarn Jarujareet Kitiporn Plaimas Parvapan Bhattarakosol Suthiluk Patumraj Wibool Piyawattanametha |
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| Affiliation: | 1. NECTEC, National Science and Technology Development Agency (NSTDA), Pathumthani, Thailand;2. Advanced Imaging Research Center, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand;3. Center of Research in Optoelectronics and Communications and Control Systems (BU‐CROCCS), Bangkok University, Pathumthani, Thailand;4. Advanced Virtual and Intelligent Computing Research Center (AVIC), Faculty of Science, Chulalongkorn University, Bangkok, Thailand;5. Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand;6. Department of Physiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand;7. Department of Electronics, Faculty of Engineering, King Mongkut's Institute of Technology Ladkrabang, Bangkok, Thailand |
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| Abstract: | Protein p16INK4a (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. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd. |
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| Keywords: | p16 cervical cancer phage display peptide‐protein docking surface alignment |
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