Novel p16 binding peptide development for p16-overexpressing cancer cell detection using phage display

J Pept Sci. 2015 Apr;21(4):265-73. doi: 10.1002/psc.2726. Epub 2015 Mar 10.


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.

Keywords: cervical cancer; p16; peptide-protein docking; phage display; surface alignment.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Cyclin-Dependent Kinase Inhibitor p16 / chemistry*
  • Humans
  • Molecular Docking Simulation
  • Neoplasms / diagnosis*
  • Neoplasms / metabolism
  • Peptide Library*
  • Protein Binding


  • Cyclin-Dependent Kinase Inhibitor p16
  • Peptide Library