Screening of a Specific Peptide Binding to VPAC1 Receptor from a Phage Display Peptide Library

Abstract

Background/PurposeThe VPAC1 receptor, a member of the vasoactive intestinal peptide receptors (VIPRs), is overexpressed in the most frequently occurring malignant tumors and plays a major role in the progression and angiogenesis of a number of malignancies. Recently, phage display has become widely used for many applications, including ligand generation for targeted imaging, drug delivery and therapy. In this work, we developed a panning procedure using a phage display peptide library to select a peptide that specifically binds to the VPAC1 receptor to develop a novel targeted probe for molecular imaging and therapy.MethodsCHO-K1 cells stably expressing VPAC1 receptors (CHO-K1/VPAC1 cells) were used to select a VPAC1-binding peptide from a 12-mer phage peptide library. DNA sequencing and homologous analysis of the randomly selected phage clones were performed. A cellular ELISA was used to determine the most selectively binding peptide for further investigation. Binding specificity to the VPAC1 receptor was analyzed by competitive inhibition ELISA and flow cytometry. The binding ability of the selected peptide to CHO-K1/VPAC1 cells and colorectal cancer (CRC) cell lines was confirmed using fluorescence microscopy and flow cytometry.ResultsA significant enrichment of phages that specifically bound to CHO-K1/VPAC1 cells was obtained after four rounds of panning. Of the selected phage clones, 16 out of 60 shared the same peptide sequence, GFRFGALHEYNS, which we termed the VP2 peptide. VP2 and vasoactive intestinal peptide (VIP) competitively bound to the VPAC1 receptor. More importantly, we confirmed that VP2 specifically bound to CHO-K1/VPAC1 cells and several CRC cell lines.ConclusionOur results demonstrate that the VP2 peptide could specifically bind to VPAC1 receptor and several CRC cell lines. And VP2 peptide may be a potential candidate to be developed as a useful diagnostic molecular imaging probe for early detection of CRC.

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