Screening and Identification of Small Peptides Targeting Fibroblast Growth Factor Receptor2 using a Phage Display Peptide Library

Summary

Herein, we present a detailed protocol for screening small peptides that bind to FGFR2 using a phage display peptide library. We further analyze the affinity of the selected peptides toward FGFR2 in vitro and its ability to suppress cell proliferation.

Abstract

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various biological activities including cell proliferation, survival, migration and differentiation. Several aberrations in the FGFR signaling pathway, owing to mutations or gene amplification events, have been identified in various types of cancers. Hence, recent research has focused on developing strategies involving therapeutic targeting of FGFRs. Current FGFR inhibitors at various stages of pre-clinical and clinical development include either small molecule inhibitors of tyrosine kinases or monoclonal antibodies, with only a few peptide- based inhibitors in the pipeline. Here, we provide a protocol using phage display technology to screen small peptides as antagonists of FGFR2. Briefly, a library of phage-displayed peptides was incubated in a plate coated with FGFR2. Subsequently, unbound phage was washed off by TBST (TBS + 0.1% [v/v] Tween-20), and bound phage was eluted with 0.2 M glycine-HCl buffer (pH 2.2). The eluted phage was further amplified and used as input for the next round of biopanning. Following three rounds of biopanning, the peptide sequences of individual phage clones were identified by DNA sequencing. Finally, the screened peptides were synthesized and analyzed for affinity and biological activity.

Introduction

Fibroblast growth factor receptors (FGFRs) play key roles in cell proliferation, wound healing, and angiogenesis in vivo¹. Aberrant activation of FGFR signaling observed in a variety of tumors^2,3,4,5 includes gene amplification, gene mutations, chromosomal aberrations, and excessive ligand secretion⁶. Many inhibitors targeting FGFRs have shown promising therapeutic effects in clinical trials and are mainly classified into three types: (1) small molecule kinase inhibitors, which bind to the intr....

Protocol

1. Reagent preparation

1. LB (lysogeny broth) medium: Dissolve 1 g of tryptone, 0.5 g of yeast extract, and 0.5 g of NaCl in 100 mL of H₂O. Autoclave and store at 4 °C.
2. Tetracycline stock: Prepare 20 mg/mL in 1:1 ethanol:H₂O. Store at -20 °C, and vortex before using.
3. IPTG/X-gal solution: Mix 0.5 g of IPTG (isopropyl β-D-1-thiogalactopyranoside) and 0.4 g of X-gal (5-bromo-4-chloro-3-indolyl β-D-galactoside) in 10 mL of DMF (dimethyl formamide). The solution can be stored at -20 °C for one year in the dark.
4. LB + Tetracycline (Tet) plates: Dissove 1 g of bacto-tryptone, 0.5 g of yeast ....

Results

Obtaining a high affinity small peptide targeting FGFR2.

To screen phages targeting FGFR2, a Ph.D.-7 library was used in this study. A schematic representation of the workflow is shown in Figure 1. In this process, the number of phage input (PFU) was kept unchanged, whereas the coating concentration of FGFR2 protein was reduced gradually. Results of the phage titer suggested that the number of recovered phages increased gradually, and after 3 roun.......

Discussion

A combinatorial phage library is a powerful and effective tool for high-throughput screening of novel peptides that can bind target molecules and regulate their function¹³. Currently, phage display peptide libraries have a wide range of applications. For example, they can be used for selecting bioactive peptides bound to receptor proteins²³, non-protein targets^24,25, disease-specific antigen mimics^.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22 μm Filter	Merck Millipore	MPGP002A1
35 cm2 Small dish	Thermo	150460
70% Ethanol	Guangzhou chemical reagent factory	64-17-5
-96 gIII sequencing primer			Synthesis from Sangon Biotech (Shanghai) Co., Ltd.
96-well plate	Nest	701001-2
Agar	Beyotime	ST004D
Bacto-Tryptone	Oxoid	L0037
BALB/c 3T3 cells	ATCC	CRL-­6587
BSA	Biodragon	BD-M10110
CCK-8 kit	DOJINDO	CK04
DMEM	Hyclone	sh30243.01
DMF	Newprobe	PB10247
EDTA	Invitrogen	15576028
FGF2 Protein	Sino Biological Inc.	10014-HNAE	Purity >95%
Glycine	Sigma	G8898-1KG
IPTG	Beyotime	ST097
ITC200 system	MicroCal Omega
NaCl	Sigma	S6191
NaHCO3	Guangzhou chemical reagent factory	144-55-8
NaI	Bidepharm	BD40879
NaOH	Guangzhou chemical reagent factory	1310-73-2
PEG–8000	Sigma	P2139-250
Ph.D.-7 phage display peptide library kit	New England BioLabs	E8100S	Containing the Ph.D.-7 phage library, E. coli ER2738 host strain and M13KE control phage
Recombinant FGFR2 extracellular domain proteins	Sino Biological Inc.	10824-H08H	Purity > 97%
Small peptide			Synthesis from GL Biochem Ltd. (Shanghai, China)
Tetracycline	Sigma	S-SHS-5
Tris	Sigma	SLF-T1503
Tween-20	Beyotime	ST825
X-gal	Beyotime	ST912
Yeast extract	Oxoid	LP0021