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.

Engineering Extracellular Vesicles to Target Pancreatic Tissue In Vivo

“…The p88 peptide, known to bind (FXYD2)γa on pancreatic β-cells, was used as a targeting ligand for pancreatic β-cells [48]. The pep1 peptide raised against p16-overexpressing cancer cells served as a control peptide with a negligible affinity for pancreatic cells in our system [63]. In order to label EVs with these peptides, we fused peptide sequences followed by the (GGGGS) 3 linker with the C1C2 domain of lactadherin (Fig.…”

Caracterização de alvos moleculares em tumores associados ao Papilomavírus Humano

“…A técnica de Peptide Phage Display permite a investigação de interações entre ligante e receptor, através da construção de um mapa de sítios de ligação do ligante ou receptor a partir das sequências de peptídeos selecionados (Giordano et al, 2001). Mais especificamente no estudo do câncer, a técnica de Peptide Phage Display tem sido amplamente utilizada em diferentes estratégias como: em modelos de inibição de fatores pró-angiogênicos como o bFGF (Yayon et al, 1993), em diagnóstico experimental de marcadores de câncer cervical como p16 (Khemthongcharoen et al, 2015) e na busca de antagonistas para reativar a via de pRB/E2F (Guo, C. P. et al, 2011).…”