Identification of an Indiscriminate Peptide by Phage Display Technology

Abstract

Phage display has been widely used to develop high‐affinity peptide ligands against proteins and cell lines. Such peptides are typically selected using biopanning to exhibit significant binding affinity and specificity against a molecular target. Rarely are the same peptides identified from separate phage display selections, since the presence of these ligands in a selection typically signify specific binding to the target. However, here, we report the identification of the same peptide (TG1) from two different phage display selections against the hemagglutinin (HA) epitope and human pancreatic cancer (Mia Paca‐2) cells.Phage display selections were conducted to identify a peptide with high affinity and specificity for the HA epitope. In brief, a pre‐cleared 15‐mer fUSE5 phage library was subjected to four rounds of selections against 0.04–4 μg HA. Following the last round of selection, the phage DNA was amplified using PCR and submitted to next‐generation sequencing. These results identified peptide TG1 (RNVPPIFNDVYWIAF) as the most prevalent ligand in the selection. Modified enzyme‐linked immunosorbent assay (ELISA) results showed that 109 nM TG1 bound significantly higher (p<0.05) to 5 μg immobilized HA compared to a non‐relevant peptide J18 known to bind ovarian adenocarcinoma (SKOV‐3) cells. In a separate, previously reported phage display selection, the same 15‐mer fUSE5 phage library was subjected to selections against human pancreatic cancer (Mia‐Paca‐2) cells. Again, next‐generation sequencing results identified TG1 as the third most prevalent peptide. Modified ELISA results using 10 μM TG1, and a known pancreatic cancer‐targeting peptide MCA2, showed that both ligands bound significantly (p<0.01) to Mia‐Paca‐2 cells compared to dimethyl sulfoxide (DMSO; vehicle). A literature search revealed that TG1 was identified by two separate research groups conducting phage display selections against caveolin protein and mouse embryos. These results indicate that the peptide is an indiscriminate binder. Thus, we sought to elucidate the binding properties of peptide TG1 and phage TG1 (pTG1).Modified ELISA with 10 μM TG1, MCA2, J18, or DMSO were carried out against SKOV‐3 and human normal pancreatic (hTERT‐HPNE) cells. Results showed that TG1 bound significantly higher (p<0.01) to both SKOV‐3 and hTERT‐HPNE cells compared to DMSO. As expected MCA2 showed no binding to either cell line. Peptide J18 showed significant binding (p<0.05) to SKOV‐3 cells.Phage qPCR analysis was performed to further elucidate the binding properties of the pTG1. 1010 V/mL pTG1, pancreatic cancer‐targeting pMCA1, or WT phage was incubated with Mia‐Paca‐2, SKOV‐3, or hTERT‐HPNE cells. After washing with Tris‐buffered saline (TBS), bound phage were eluted with 2.5% 3‐cholamidopropyl dimethylammonio 1‐propanesulfonate (CHAPS). Collected phage virions (5 μL) were quantified by qPCR using Fast SYBR Green Master Mix on an Applied Biosystems StepOnePlus Real‐Time qPCR System (Applied Biosystems, CA). The results revealed that pTG1 demonstrated significantly higher binding (p<0.01) compared to WT for all cell lines. As expected, pMCA1 showed binding to Mia‐Paca‐2, and pJ18 exhibited binding to SKOV‐3 cells. Taken together, these results indicate that TG1 indiscriminately binds multiple targets.