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 intracellular domain of FGFR, (2) antagonists targeting the extracellular segment, and (3) FGF ligand traps⁶. Although several of the small molecule kinase inhibitors have good therapeutic effects both in vitro and in vivo⁷, most of them have poor target specificity and show adverse effects such as hypertension⁸. The majority of the antagonists are monoclonal antibodies^9,10 and polypeptides¹¹. Peptides have advantages over small molecules due to their specificity and lower side effects. They also retain cell permeability and do not accumulate in specific organs as compared to protein drugs¹². Hence, targeted small peptides are both effective and prospective therapeutic agents.

Phage display technology is an easy but powerful tool for identifying small peptides which can bind to a given molecule^13,14,15. We used a phage display peptide library that is based on a simple M13 phage with over 10⁹ different peptide sequences displayed at the tail for binding to the target molecule (see Table of Materials)¹⁶. Due to the high affinity of phages towards the given molecule, unbound phages can be washed away, and only tightly bound phages with the desired short peptides are retained. The given molecular targets can be immobilized proteins^17,18, carbohydrates, cultured cells, or even inorganic materials^19,20. An exciting case was reported where organ-specific peptides were selected in vivo using phage display technology²¹. The advantages of phage display technology include high throughput, ease of operation, low cost and a wide range of applications²².

In this study, we provide a detailed protocol of screening small peptides binding to the immobilized protein (FGFR2) using a phage display library. The efficacy of the technology is also examined by measuring the affinity of the obtained peptide towards FGFR2 by Isothermal Titration Calorimetry (ITC), and the biological activity by a cell proliferation assay. The method may be extended for screening small peptides that bind to carbohydrates, cultured cells, or even inorganic materials.

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 extract, 0.5 g of NaCl, and 1.5 g of agar in 100 mL of H₂O. Autoclave and cool to < 70 °C. Add 100 μL of the tetracycline stock and pour plates. Store plates at 4 °C in the dark.
5. LB + IPTG/X-gal plates: Dissolve 1 g of bacto-tryptone, 0.5 g of yeast extract, 0.5 g of NaCl, and 1.5 g of agar in 100 mL of H₂O. Autoclave and cool to < 70°C. Add 100 μL of the IPTG/X-gal solution and pour plates. Store plates at 4 °C in the dark.
6. Glycine-HCI buffer (0.2 M): Dissolve 1.5014 g of glycine and 100 mg of BSA (bovine serum albumin) in 100 mL of H₂O and adjust the pH to 2.2 with HCl. Filter the solution using a 0.22 μm filter and store at 4 °C.
7. Tris-HCl buffer (1 M): Dissolve 12.11 g of Tris (tris(hydroxymethyl)aminomethane) in 100 mL of H₂O and adjust the pH to 9.1 with HCl. Autoclave and store at 4 °C.
8. Coating buffer (0.1 M): Dissolve 1.68 g of NaHCO₃ in 200 mL of H₂O and adjust the pH to 8.6 with NaOH. Filter the solution using a 0.22 μm filter and store at 4 °C.
9. Blocking buffer: Prepare 0.1 M NaHCO₃ (pH 8.6) with 5 mg/mL BSA. Filter the solution using a 0.22 μm filter and store at 4 °C.
10. TBS: Prepare 50 mM Tris-HCl (pH 7.5) with 150 mM NaCl. Autoclave and store at room temperature.
11. 0.05%, 0.1%, or 0.5% TBST: Prepare TBS with 0.05%, 0.1%, or 0.5% (v/v) Tween-20. Filter using a 0.22 μm filter and store at room temperature.
12. PEG/NaCl: Prepare 20% (w/v) polyethylene glycol–8000 with 2.5 M NaCl. Autoclave and store at room temperature.
13. TE solution: Mix 1 mL of 1 M Tris-HCl (pH8.0) and 2 mL of 50 mM EDTA (pH 8.0) and adjust the volume to 100 mL with H₂O. Autoclave and store at room temperature.
14. Iodide buffer: Dissolve 30 g of NaI in 50 mL of TE, pH 8.0. Store the solution at room temperature in the dark.
15. Top Agar: Dissolve 1 g of tryptone, 0.5 g of yeast extract, 0.5 g of NaCl, and 0.7 g agarin in 100 mL of H₂O. Autoclave and dispense into 15 mL aliquots. Store at room temperature for use.

2. First round of biopanning: Screening phage clones that bind to the extracellular domain of FGFR2

NOTE: Use aerosol-resistant pipette tips and wear gloves for all protocols in order to minimize contamination with environmental bacteriophages.

1. Prepare a 10 µg/mL FGFR2 (Purity > 97%; see Table of Materials) solution in coating buffer. Add 1 mL of the prepared solution to a 35 cm² dish and swirl repeatedly until the surface is completely wet. Incubate overnight at 4 °C with shaking.
   NOTE: To avoid selection of decoy target binders, the target protein must be highly purified.
2. Inoculate 5 mL of LB + Tet medium with Escherichia coli (E. coli) ER2738 for 5–10 hours at 37 °C with shaking. The amplified ER2738 cells will be used for titration in step 3.3.
3. Pour off the coating solution from the dish (the excess solution must be removed) and add the blocking solution. Incubate at 4 °C for at least 1 h.
4. Discard the blocking solution and wash 6 times quickly with TBST (TBS + 0.05% (v/v] Tween-20). Avoid drying out the dish.
5. Reconstitute the original phage library in 1 mL of TBST [final concentration of 10¹¹ plaque forming units (PFU)], add to the coated dish, and rock gently for 2 h on a shaker at room temperature.
6. Discard the supernatant and wash the dish 10x with TBST (TBS + 0.05% (v/v) Tween-20), as performed in step 2.4.
   NOTE: The unbound phages must be removed thoroughly by vigorous washing.
7. Elute the bound phage by adding 1 mL of 0.2 M glycine-HCl buffer (pH 2.2) to the dish and rocking gently for 10 min at room temperature.
8. Transfer the eluate into a sterilized microfuge tube and neutralize it with 100 µL of 1 M Tris-HCl, pH 9.1.
   NOTE: In step 2.6, remove the unbound phage as much as possible by washing the dish 10x vigorously. This is a crucial step. The eluted phage can be stored at 4 °C for one week. However, it is best to go forward to the next step as soon as possible.

3. Titer determination of the eluted phages

1. Pre-warm LB + IPTG/X-gal plates for at least 1 h at 37 °C before use.
2. Prepare 100 µL of 10-fold serial dilutions (i.e., 10¹, 10², 10³ and 10⁴) of the eluate from step 2.8 in LB. Use tips with filter cartridges to avoid contamination.
3. Let the culture from step 2.2 reach mid-log phase (OD600 ≈ 0.5). Dispense 200 µL of the culture into sterilized microfuge tubes, one for each eluate dilution.
4. To initiate the infection, add 10 µL of each dilution from step 3.2 to individual microfuge tubes containing the E. coli ER2738 culture from step 3.3. Vortex quickly and incubate at room temperature for 5 min.
5. Coat 90 μL of the mixture from step 3.4 into the LB +IPTG/X-gal plates with coating sticks.
6. Optionally, melt top agar in a microwave, dispense 3 mL into sterile culture tubes, one for each eluate dilution, and maintain tubes at 45 °C. Transfer the mixture from step 3.4 to culture tubes containing 45 °C top agar. Mix quickly and immediately pour the culture onto a pre-warmed LB/IPTG/X-gal plate. Gently tilt and rotate the plate to spread the top agar evenly.
7. After 5 min, invert and incubate plates overnight at 37 °C.
8. Count plaques on plates with approximately 100 plaques. Multiply each number by the dilution factor for that plate to get the phage titer in PFU per 10 µL.
9. Inoculate another 5 mL of LB + Tet medium with E. coli ER2738 at 37 °C overnight with shaking for phage amplification.
   NOTE: Use the culture of ER2738 in mid-log phase (OD600 ≈ 0.5) for phage titer. If most of the plaques are white on X-gal/IPTG plates, it means that the pool of phage has been contaminated with wild bacteriophages. If contamination occurs, it is necessary to start again with the panning steps from the uncontaminated phage stock solution.

4. Phage amplification

1. Dilute (1:100) the overnight culture from step 3.8 in 20 mL of LB in a 250 mL Erlenmeyer flask. Add the unamplified eluate and incubate with vigorous shaking for 4.5 h at 37 °C.
2. Transfer the culture to a centrifuge tube and centrifuge for 10 min at 12,000 x g at 4 °C. Transfer the supernatant to a fresh tube, centrifuge again, and discard the pellet.
3. Transfer the upper 80% of the supernatant to a fresh tube and add to it 1/6 volume of 20% PEG/2.5 M NaCl. Allow the phage to precipitate at 4 °C overnight.
   NOTE: Mix the culture supernatant with PEG/2.5 M NaCl well.
4. Centrifuge the precipitated phage at 12,000 x g for 15 min at 4 °C. Discard the supernatant, centrifuge again, and remove the residual supernatant with a pipette.
5. Resuspend the pellet in 1 mL of TBS and transfer the supernatant to a microfuge tube. Centrifuge at 12,000 x g for 5 min at 4 °C.
6. Transfer the supernatant to a fresh microfuge tube and precipitate again by adding 1/6 volume of 20% PEG/2.5 M NaCl. Incubate on ice for 15–60 min. Centrifuge at 12,000 x g for 10 min at 4 °C. Discard the supernatant, re-spin, and remove the residual supernatant with a pipette.
7. Resuspend the pellet in 200 μL of TBS and centrifuge for an additional minute to remove impurities. Transfer the supernatant to a fresh microfuge tube to obtain the amplified eluate.
8. Titer the amplified phage as described in section 3.
   NOTE: The dilution of the amplified eluate is higher compared to the unamplified eluate; serial dilutions of 10⁸–10¹¹ are typically suggested.

5. Second round of biopanning

1. Repeat sections 2 to 4.
2. Titer the second round of the amplified eluate on LB + IPTG/X-gal plates as described in section 3.
   NOTE: Reduce the coating concentration of FGFR2 protein to 5 μg/mL. Use the amplified phage from step 4.7 as the input and keep the titer same as in the first round (10¹¹ PFU). Shorten the incubation time of phage and FGFR2-coated dish to 1 h and increase the Tween concentration to 0.1% (v/v) in the wash steps.

6. Third round of biopanning

1. Repeat steps 2.1-2.6.
2. Elute the bound phage by adding 1 mL of 20 μM bFGF solution (a ligand for FGFR2) to the dish and rocking gently for 60 min at room temperature.
3. Repeat steps 3.1-3.7.
   NOTE: Reduce the coating concentration of the FGFR2 protein to 2.5 μg/mL. Use the second round of amplified phage from section 5 as the input and keep the titer same as in the first round (10¹¹ PFU). Shorten the incubation time of phage and FGFR2-coated dish to 30 min and increase the Tween concentration to 0.5% (v/v) in the wash steps.

7. Acquisition of plaque DNA for sequencing

1. Plaque amplification
   1. Dilute (1:100) an overnight culture of ER2738 in LB. Dispense 1 mL of diluted culture into a 2 mL tube, one for each clone to be characterized.
   2. Use a pipette tip to pick one well-separated blue colored plaque from a titer plate from section 6 and transfer to the tube containing the diluted culture. Pick a total of 15 plaques.
      NOTE: Plates should be < 1–3 days old, stored at 4 °C, and have < 100 plaques.
   3. Incubate the tubes with vigorous shaking for 4.5 h at 37 °C.
   4. Transfer the cultures to centrifuge tubes and centrifuge at 12,000 x g for 30 s. Transfer the supernatant to a fresh tube and centrifuge again.
   5. Using a pipette, transfer the upper 80% of the supernatant to a fresh tube. Label this as the amplified phage stock.
      NOTE: Dilute the amplified phage with an equal volume of 100% sterile glycerol and store at -80 °C for later use.
2. Extraction of plaque DNA
   1. Transfer 500 µL of the amplified phage to a fresh microfuge tube and add a 1/6 volume of 20% PEG/2.5 M NaCl. Invert to mix and let it stand for 10–20 min at room temperature.
   2. Centrifuge at 12,000 x g for 10 min at 4 °C and then pour off the supernatant.
      NOTE: Phage pellet may not be visible.
   3. Centrifuge again. Carefully remove any remaining supernatant.
   4. Resuspend the pellet thoroughly in 100 µL of the iodide buffer by vigorously tapping the tube.
   5. Add 250 µL of 100% ethanol and incubate for 15 min at room temperature.
   6. Centrifuge at 12,000 x g for 10 min at 4 °C and remove the supernatant. Wash the pellet with 0.5 mL of 70% ethanol, quickly centrifuge again, remove the supernatant, and briefly dry the pellet.
   7. Resuspend the pellet in 30 µL of TE buffer and use this for sequencing.
      NOTE: Pre-cool the 70% ethanol at -20°C in advance of Step 7.2.6. The phage pellet must be thoroughly resuspended in the iodide buffer prior to the addition of ethanol.

8. Identification of the peptide sequence

1. Use the -96 gIII sequencing primer (5´-CCC TCA TAG TTA GCG TAA CG –3´) for sequencing.
2. Analyze the peptide sequences displayed on the phage based on the DNA sequencing results.
3. Synthesize small peptides and analyze by high-performance liquid chromatography (HPLC) and mass spectrometry to confirm their purity (≥98%).

9. Detecting the affinity of the obtained small peptide and extracellular protein of FGFR2 by ITC

1. Degas sterile water using an ultrasonic instrument. Dissolve the small peptide and the FGFR2 extracellular protein in sterile water. Centrifuge these samples to remove residual precipitate.
2. Perform the ITC titration experiments at 25 °C. Titrate 40 μM of small peptide in the cell with 1.5 μM of FGFR2 protein from the syringe, with a stirring speed of 750 rpm. Add the FGFR2 protein in 2 μL aliquots (for a total of 19 injections) at 5-min intervals.
3. Analyze the ITC data using a single-site binding model.
   NOTE: Sterile water must be degassed, and all samples must be centrifuged to remove air bubbles and residual impurities. The experiment must be conducted at a constant temperature of 25 °C.

10. Verifying the biological activity of the obtained small peptide using a cell proliferation assay

1. Seed BALB/c 3T3 cells (3.0 × 10³ cells/well, 100 μL) in a 96-well plate with Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) overnight at 37 °C and 5% CO₂.
2. Replace the supernatant with fresh DMEM with 0.5% FBS and starve the cells for 12 h.
3. Make serial dilutions of the small peptide (0, 0.0064, 0.032, 0.16, 0.8, 4, 20, and 100 μM) with fresh DMEM supplemented with 0.5% FBS (6 duplicate wells /concentration). Remove the supernatant from the 96-well plate and add 100 μL of the small peptide solution per well. Incubate for 48 h at 37 °C and 5% CO₂.
4. Replace the supernatant with fresh DMEM containing 10% Cell Counting Kit (CCK)-8 solution and incubate for 2 h.
5. Measure the absorbance of each well at 450 nm with a microplate reader and analyze cell viability.

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