This work was supported by the Science and Technology Program of Guangzhou (No. 2016201604030039).

1. Reagent preparation
<ol>
	<li>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 &#176;C.</li>
	<li>Tetracycline stock: Prepare 20 mg/mL in 1:1 ethanol:H2O. Store at -20 &#176;C, and vortex before using.</li>
	<li>IPTG/X-gal solution: Mix 0.5 g of IPTG (isopropyl &#946;-D-1-thiogalactopyranoside) and 0.4 g of X-gal (5-bromo-4-chloro-3-indolyl &#946;-D-galactoside) in 10 mL of DMF (dimethyl formamide). The so...

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M., Highsmith, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage+display+technology:+clinical+applications+and+recent+innovations.">Phage display technology: clinical applications and recent innovations.</a> Clinical Biochemistry. 35, 425-445 (2002).</li><li>Pasqualini, R., Ruoslahti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+targeting+In+vivo+using+phage+display+peptide+libraries.">Organ targeting In vivo using phage display peptide libraries.</a> Nature. 380 (6572), 364-366 (1996).</li><li>Liu, R., Li, X., Xiao, W., Lam, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-targeting+peptides+from+combinatorial+libraries.">Tumor-targeting peptides from combinatorial libraries.</a> Advanced Drug Delivery Reviews. 110-111, 13-37 (2017).</li><li>Binetruy-Tournaire, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+peptide+blocking+vascular+endothelial+growth+factor+(VEGF)-mediated+angiogenesis.">Identification of a peptide blocking vascular endothelial growth factor (VEGF)-mediated angiogenesis.</a> The EMBO Journal. 19, 1525-1533 (2000).</li><li>Peng, Y., Zhang, Y., Mitchell, W. J., Zhang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Lipopolysaccharide-Targeted+Peptide+Mimic+Vaccine+against+Q+Fever.">Development of a Lipopolysaccharide-Targeted Peptide Mimic Vaccine against Q Fever.</a> Journal of Immunology. 189, 4909-4920 (2012).</li><li>Lamichhane, T. N., Abeydeera, N. D., Duc, A. C., Cunningham, P. R., Chow, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+Peptides+Targeting+Helix+31+of+Bacterial+16S+Ribosomal+RNA+by+Screening+M13+Phage-Display+Libraries.">Selection of Peptides Targeting Helix 31 of Bacterial 16S Ribosomal RNA by Screening M13 Phage-Display Libraries.</a> Molecules. 16, 1211-1239 (2011).</li><li>Sahin, D., Taflan, S. O., Yartas, G., Ashktorab, H., Smoot, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+and+Identification+of+Peptides+Specifically+Targeted+to+Gastric+Cancer+Cells+from+a+Phage+Display+Peptide+Library.+Asian+Pacific.">Screening and Identification of Peptides Specifically Targeted to Gastric Cancer Cells from a Phage Display Peptide Library. Asian Pacific.</a> Journal of Cancer Prevention. 19 (4), 927-932 (2018).</li><li>Kelly, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+nanoparticles+for+imaging+incipient+pancreatic+ductal+adenocarcinoma.">Targeted nanoparticles for imaging incipient pancreatic ductal adenocarcinoma.</a> PLOS Medicine. 5 (4), e85 (2008).</li><li>Sugihara, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+pro-apoptotic+peptides+as+potential+therapy+for+peritoneal+endometriosis.">Development of pro-apoptotic peptides as potential therapy for peritoneal endometriosis.</a> Nature Communications. 5, 4478 (2014).</li><li>Rafii, S., Avecilla, S. T., Jin, D. 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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 mimics...

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.

<table>
	<tbody>
		<tr>
			<td>0.22 &#956;m Filter</td>
			<td>Merck Millipore</td>
			<td>MPGP002A1</td>
			<td></td>
		</tr>
		<tr>
			<td>35 cm2 Small dish</td>
			<td>Thermo</td>
			<td>150460</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>Guangzhou chemical reagent factory</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>-96 gIII sequencing primer</td>
			<td></td>
			<td></td>
			<td>Synthesis from Sangon Biotech (Shanghai) Co., Ltd.</td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Nest</td>
			<td>701001-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Beyotime</td>
			<td>ST004D</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto-Tryptone</td>
			<td>Oxoid</td>
			<td>L0037</td>
			<td></td>
		</tr>
		<tr>
			<td>BALB/c 3T3 cells</td>
			<td>ATCC</td>
			<td>CRL-&#173;6587</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Biodragon</td>
			<td>BD-M10110</td>
			<td></td>
		</tr>
		<tr>
			<td>CCK-8 kit</td>
			<td>DOJINDO</td>
			<td>CK04</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Hyclone</td>
			<td>sh30243.01</td>
			<td></td>
		</tr>
		<tr>
			<td>DMF</td>
			<td>Newprobe</td>
			<td>PB10247</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Invitrogen</td>
			<td>15576028</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2 Protein</td>
			<td>Sino Biological Inc.</td>
			<td>10014-HNAE</td>
			<td>Purity &#62;95%</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma</td>
			<td>G8898-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Beyotime</td>
			<td>ST097</td>
			<td></td>
		</tr>
		<tr>
			<td>ITC200 system</td>
			<td>MicroCal Omega</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S6191</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Guangzhou chemical reagent factory</td>
			<td>144-55-8</td>
			<td></td>
		</tr>
		<tr>
			<td>NaI</td>
			<td>Bidepharm</td>
			<td>BD40879</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Guangzhou chemical reagent factory</td>
			<td>1310-73-2</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG&#8211;8000</td>
			<td>Sigma</td>
			<td>P2139-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Ph.D.-7 phage display peptide library kit</td>
			<td>New England BioLabs</td>
			<td>E8100S</td>
			<td>Containing the Ph.D.-7 phage library, E. coli ER2738 host strain and M13KE control phage</td>
		</tr>
		<tr>
			<td>Recombinant FGFR2 extracellular domain proteins</td>
			<td>Sino Biological Inc.</td>
			<td>10824-H08H</td>
			<td>Purity &#62; 97%</td>
		</tr>
		<tr>
			<td>Small peptide</td>
			<td></td>
			<td></td>
			<td>Synthesis from GL Biochem Ltd. (Shanghai, China)</td>
		</tr>
		<tr>
			<td>Tetracycline</td>
			<td>Sigma</td>
			<td>S-SHS-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma</td>
			<td>SLF-T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Beyotime</td>
			<td>ST825</td>
			<td></td>
		</tr>
		<tr>
			<td>X-gal</td>
			<td>Beyotime</td>
			<td>ST912</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Oxoid</td>
			<td>LP0021</td>
			<td></td>
		</tr>
	</tbody>
</table>

screening and identification of small peptides targeting fibroblast growth factor receptor2 using a phage display peptide library

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.

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...

Watch this Scientific Journal Video about Screening and Identification of Small Peptides Targeting Fibroblast Growth Factor Receptor2 using a Phage Display Peptide Library at JoVE.com

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

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 ...

Compared with other methods, this protocol is easier to find out new antagonisters, agonisteres, and allosteric modulators quantitatively and qualitatively without knowing the hydro-dilution structure and formation of the target protein. This protocol is high efficient, easy to handle, economical, and commercially available. Combine this high throughput screening method with ITC analysis, the functional small peptides can be obtained quantitatively and qualitatively.Demonstrating the procedure will be Ying Zhao and Qiang Wang, graduate students from our laboratory. To begin this procedure, prepare a solution of FGFR2 in coating buffer at a concentration of 10 micrograms per milliliter. Add one milliliter of the solution to a 35 square centimeter dish and swirl repeatedly until the surface is completely wet.Incubate overnight at four degrees Celsius with shaking. The next day, pour off the coating solution and add the prepared blocking solution. Incubate at four degrees Celsius for at least one hour, then discard the blocking solution and quickly wash the plate six times with TBST while making sure the dish does not dry out.Reconstitute the original phage library in one milliliter of TBST and add this to the coated dish. Gently rock the plate on a shaker at room temperature for two hours. After this, discard the supernatant and wash the dish 10 times with TBST.Elute the bound phage by adding one milliliter of 0.2 molar glycine HCL to the dish and rocking gently at room temperature for 10 minutes. Transfer the eluent into a sterilized microcentrifuge tube and neutralize it with 100 microliters of one molar Tris-HCL at pH 9.1. First, prewarm the prepared LB and IPTG X-Gal plates at 37 degrees Celsius for at least one hour before use.Using pipette tips with filter cartridges, prepare 100 microliters of tenfold dilutions of the eluent in LB.After the prepared E.coli culture reaches mid-log phase, dispense 200 microliters of the culture into sterilized microcentrifuge tubes one for each eluent dilution. To initiate the infection, add 50 microliters of each dilution to the tubes containing the E.coli culture. Vortex quickly and incubate at room temperature for five minutes.Then use coating sticks to coat the LB and IPTG X-GAL plates with 90 microliters of this mixture. After five minutes, invert the plates and incubate them overnight at 37 degrees Celsius. Count plaques when they appear.To begin, dilute the overnight culture in 20 milliliters of LB in a 250 milliliter Erlenmeyer flask. Add the unamplified eluent and incubate at 37 degrees Celsius with vigorous shaking for 4-1/2 hours. Next, centrifuge the culture at 12, 000 times G and four degrees Celsius for 10 minutes.Transfer the supernatant to a fresh tube. Centrifuge again using the same conditions and discard the pellet. Transfer the upper 80%of the supernatant to a fresh tube and add it to 1/6th volume of 20%polyethylene glycol and 2.5 molar sodium chloride.Allow the phage to precipitate overnight at four degrees Celsius. The next day, centrifuge the precipitated phage at 12, 000 times G and four degrees Celsius for 15 minutes. Discard the supernatant and centrifuge again using the same conditions.Use a pipette to remove the residual supernatant. Resuspend the pellet in one milliliter of TBS and transfer the supernatant to a microcentrifuge tube. Centrifuge at 12, 000 times G and four degrees Celsius for five minutes.Transfer the supernatant to a fresh microcentrifuge tube and precipitate again by adding 1/6th volume of 20%polyethylene glycol and 2.5 molar sodium chloride. Incubate on ice for 15 to 60 minutes. Next, centrifuge at 12, 000 times G and four degrees Celsius for 10 minutes.Discard the supernatant and centrifuge again using the same conditions. Use a pipette to remove the residual supernatant. Resuspend the pellet in 200 microliters of TBS and centrifuge for an additional minute to remove impurities.Then transfer the supernatant to a fresh microcentrifuge tube to obtain the amplified eluent. In this study, the number of phage input is kept unchanged whereas the coating concentration of FGFR2 protein is gradually reduced. Results of the phage titer suggest that the number of recovered phages gradually increases and that after three rounds, there is a 65-fold increase as compared to that of the first round.An ITC experiment is then conducted to measure the affinity of the small peptide to FGFR2. The results indicate that the SP1 peptide has high affinity towards FGFR2. This data demonstrates the efficiency of the screening protocol.To investigate the biological activity of the SP1 peptide, a fibroblast proliferation assay is performed using a CCK-8 kit. BALB/C3T3 are incubated with the SP1 peptide at different concentrations. The results suggest that the SP1 peptide suppresses the growth of the cells.The contamination by wild-type phages has to be avoided here. Make sure to use aerosol-resistant pipette tips. While growth by obtaining phage amplification.This procedure can also be used in screening small peptides that bind to carbohydrates, culture cells, and tissues. Those peptides can be further amplification over diagnostic and targeted

screening-identification-small-peptides-targeting-fibroblast-growth

Research

JoVE Journal

Bioengineering

7.4K vistas.  Guangdong Provincial Key Laboratory of Bioengineering Medicine. En comparación con otros métodos, este protocolo es más fácil de encontrar nuevos antagonistas, agonisteres y moduladores alotéricos cuantitativa y cualitativamente sin conocer la estructura hidro-dilución y la formación de la proteína diana. Este protocolo es altamente eficiente, fácil de manejar, económico y disponible comercialmente. Combinando este método de cribado de alto rendimiento con el análisis ITC, los pequeños péptidos funcionales se pueden obtener cuantitativa y cu...