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.
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.
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 tumors2,3,4,5 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 antibodies9,10 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 molecule13,14,15. 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 proteins17,18, carbohydrates, cultured cells, or even inorganic materials19,20. 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.
1. Reagent preparation
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.
3. Titer determination of the eluted phages
4. Phage amplification
5. Second round of biopanning
6. Third round of biopanning
7. Acquisition of plaque DNA for sequencing
8. Identification of the peptide sequence
9. Detecting the affinity of the obtained small peptide and extracellular protein of FGFR2 by ITC
10. Verifying the biological activity of the obtained small peptide using a cell proliferation assay
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 rounds, there was a 65-fold increase as compared to that of the first round (Table 1).
Next, we picked phage clones randomly after the third round and extracted the phage DNA for sequencing. One representative peptide (WRARVPL) obtained by screening was named SP1. It was subsequently synthesized, and its molecular weight was measured by mass spectrometry.
The SP1 peptide showed high binding affinity towards FGFR2.
An ITC experiment was conducted to measure the affinity of the small peptide to FGFR2. The result indicated that the SP1 peptide has high affinity towards FGFR2 (Kd ≈ 1.4 μM; Figure 2). This date demonstrated the efficiency of the screening protocol.
The SP1 peptide inhibited the growth of fibroblasts.
To investigate the biological activity of the SP1 peptide, a fibroblast proliferation assay was performed using a CCK-8 kit. BALB/c 3T3 cells were incubated with the SP1 peptide at different concentrations (0, 0.0064, 0.032, 0.16, 0.8, 4, 20, and 100 μM). The result suggested that the SP1 peptide suppressed the growth of BALB/c 3T3 cells (Figure 3).
Figure 1. Biopanning for small peptide targeting FGFR2 using phage display library.
(A) Schematic representation of small peptide screening using phage display library. Incubate the library containing 1011 PFU in a dish coated with FGFR2. Discard unbound phages by washing, and elute bound phages. Amplify the eluted phages and use them as input for the next round of biopanning. Please click here to view a larger version of this figure.
Figure 2. Measuring the affinity of interaction between the SP1 peptide and FGFR2 by ITC.
1.5 μM FGFR2 protein was titrated with 40 μM of the SP1 peptide. The ITC data was further analyzed using Origin 7.0 software. Please click here to view a larger version of this figure.
Figure 3. Effect of the SP1 peptide on the growth of fibroblasts.
BALB/c 3T3 cells were treated with the SP1 peptide at different concentrations (0, 0.0064, 0.032, 0.16, 0.8, 4, 20 and 100 μM). The results are expressed as the mean ± SD. Please click here to view a larger version of this figure.
Round | FGFR2 (μg) | Input phage (PFU) | Output phage (PFU) | Recovery (%) | Enrichment |
1 | 10 | 2.0 x 1011 | 3.98 x 104 | 1.99 x 10-5 | 1 |
2 | 5 | 2.0 x 1011 | 8.1 x 105 | 4.05 x 10-4 | 20 |
3 | 2.5 | 2.0 x 1011 | 2.6 x 106 | 1.3 x 10-3 | 65 |
Table 1. Enrichment of phage targeting FGFR2 relative to round 1.
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 targets24,25, disease-specific antigen mimics13, cell-specific peptides26,27, or tissue/organ-specific peptides21,28,29 and development of peptide-mediated drug delivery systems19,30. In brief, phage display peptide libraries are useful and efficient systems to identify specific peptides in basic research and development of translational medicine. In the study, a library was used for screening small peptides targeting FGFR2 for cell growth inhibition.
The critical steps of the protocol are mentioned below. In the panning step, contamination by wild-type phages must be avoided. The unbound phages must be removed thoroughly by vigorous washing. The LB + IPTG/X-gal plates for phage titer must be pre-warmed to 37 °C. For the phage titer, E. coli ER2738 must be grown till mid-log phase (OD600 ≈ 0.5). In step 4.3, the phage must be precipitated overnight at 4 °C. Well separated blue plaques must be picked from platea containing less than 100 plaques for DNA sequencing. In step 7.2.6, the 70% ethanol solution must be pre-cooled at 20 °C in advance. Lastly, for the ITC experiment, 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.
Some factors that can affect the quality of hits obtained22 are as follows: 1) In the first round of screening, the number of phage inputs needs to be 1011 PFU. However, in the next round of screening, the input can be lower. 2) A pure environment must be maintained to avoid wild-type phage contamination. 3) 3 or 4 rounds of screening are usually sufficient. Avoid over-panning the peptide library. 4) In each round, the amount of target protein is gradually reduced, whereas the content of Tween is gradually increased in the washing step. In addition, the incubation time of phage and FGFR2-coated dish is also gradually shortened. If most of the eluted phage plaques are white on X-gal/IPTG plates, this suggests contamination by wild-type phages. In order to avoid a low titer of amplified phage in the panning process, cultures must be well aerated and infected early in their growth phase (OD600 < 0.05).
In this study, the SP1 peptide showed high affinity towards FGFR2 (Kd ≈ 1.4 μM; Figure 2) and good biological activity (Figure 3). Thus, our results suggest that the protocol was effective in selecting peptides against the extracellular domain of FGFR2 protein, although note that due to the high sequence similarity of FGFR family members, the SP1 peptide may have some binding affinity with FGFR family members besides FGFR2, which could be responsible for the biological activity seen. Also, in the panning process, phage ELISA is an alternative to identify the positive phages qualitatively. However, in our lab, we always obtain the peptides by sequencing and evaluating their affinity by ITC assay quantitatively and have not had problems evaluating and selecting the candidate peptides.
The phage-display library has several advantages22. The libraries are of high capacity (up to 1011 PFU) and can be used in vitro, in vivo, and ex vivo. Libraries are highly efficient, easy to handle, inexpensive and commercially available. However, there are certain limitations of the technology. The libraries only contain proteinogenic amino acids and are only amenable to linear and simple cyclic peptides without complicated structures.
The authors have nothing to disclose.
This work was supported by the Science and Technology Program of Guangzhou (No. 2016201604030039).
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 |