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 solution can be stored at -20 &#176;C for one year in the dark.</li>
	<li>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 &#60; 70 &#176;C. Add 100 &#956;L of the tetracycline stock and pour plates. Store plates at 4 &#176;C in the dark.</li>
	<li>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 &#60; 70&#176;C. Add 100 &#956;L of the IPTG/X-gal solution and pour plates. Store plates at 4 &#176;C in the dark.</li>
	<li>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 &#956;m filter and store at 4 &#176;C.</li>
	<li>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 &#176;C.</li>
	<li>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 &#956;m filter and store at 4 &#176;C.</li>
	<li>Blocking buffer: Prepare 0.1 M NaHCO3 (pH 8.6) with 5 mg/mL BSA. Filter the solution using a 0.22 &#956;m filter and store at 4 &#176;C.</li>
	<li>TBS: Prepare 50 mM Tris-HCl (pH 7.5) with 150 mM NaCl. Autoclave and store at room temperature.</li>
	<li>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 &#956;m filter and store at room temperature.</li>
	<li>PEG/NaCl: Prepare 20% (w/v) polyethylene glycol&#8211;8000 with 2.5 M NaCl. Autoclave and store at room temperature.</li>
	<li>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.</li>
	<li>Iodide buffer: Dissolve 30 g of NaI in 50 mL of TE, pH 8.0. Store the solution at room temperature in the dark.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>Prepare a 10 &#181;g/mL FGFR2 (Purity &#62; 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 &#176;C with shaking. 
	NOTE: To avoid selection of decoy target binders, the target protein must be highly purified.</li>
	<li>Inoculate 5 mL of LB + Tet medium with Escherichia coli (E. coli) ER2738 for 5&#8211;10 hours at 37 &#176;C with shaking. The amplified ER2738 cells will be used for titration in step 3.3.</li>
	<li>Pour off the coating solution from the dish (the excess solution must be removed) and add the blocking solution. Incubate at 4 &#176;C for at least 1 h.</li>
	<li>Discard the blocking solution and wash 6 times quickly with TBST (TBS + 0.05% (v/v] Tween-20). Avoid drying out the dish.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Transfer the eluate into a sterilized microfuge tube and neutralize it with 100 &#181;L 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 &#176;C for one week. However, it is best to go forward to the next step as soon as possible.</li>
</ol>
3. Titer determination of the eluted phages
<ol>
	<li>Pre-warm LB + IPTG/X-gal plates for at least 1 h at 37 &#176;C before use.</li>
	<li>Prepare 100 &#181;L 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.</li>
	<li>Let the culture from step 2.2 reach mid-log phase (OD600 &#8776; 0.5). Dispense 200 &#181;L of the culture into sterilized microfuge tubes, one for each eluate dilution.</li>
	<li>To initiate the infection, add 10 &#181;L 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.</li>
	<li>Coat 90 &#956;L of the mixture from step 3.4 into the LB +IPTG/X-gal plates with coating sticks.</li>
	<li>Optionally, melt top agar in a microwave, dispense 3 mL into sterile culture tubes, one for each eluate dilution, and maintain tubes at 45 &#176;C. Transfer the mixture from step 3.4 to culture tubes containing 45 &#176;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.</li>
	<li>After 5 min, invert and incubate plates overnight at 37 &#176;C.</li>
	<li>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 &#181;L.</li>
	<li>Inoculate another 5 mL of LB + Tet medium with E. coli ER2738 at 37 &#176;C overnight with shaking for phage amplification. 
	NOTE: Use the culture of ER2738 in mid-log phase (OD600 &#8776; 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.</li>
</ol>
4. Phage amplification
<ol>
	<li>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 &#176;C.</li>
	<li>Transfer the culture to a centrifuge tube and centrifuge for 10 min at 12,000 x g at 4 &#176;C. Transfer the supernatant to a fresh tube, centrifuge again, and discard the pellet.</li>
	<li>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 &#176;C overnight. 
	NOTE: Mix the culture supernatant with PEG/2.5 M NaCl well.</li>
	<li>Centrifuge the precipitated phage at 12,000 x g for 15 min at 4 &#176;C. Discard the supernatant, centrifuge again, and remove the residual supernatant with a pipette.</li>
	<li>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 &#176;C.</li>
	<li>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&#8211;60 min. Centrifuge at 12,000 x g for 10 min at 4 &#176;C. Discard the supernatant, re-spin, and remove the residual supernatant with a pipette.</li>
	<li>Resuspend the pellet in 200 &#956;L of TBS and centrifuge for an additional minute to remove impurities. Transfer the supernatant to a fresh microfuge tube to obtain the amplified eluate.</li>
	<li>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&#8211;1011 are typically suggested.</li>
</ol>
5. Second round of biopanning
<ol>
	<li>Repeat sections 2 to 4.</li>
	<li>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 &#956;g/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.</li>
</ol>
6. Third round of biopanning
<ol>
	<li>Repeat steps 2.1-2.6.</li>
	<li>Elute the bound phage by adding 1 mL of 20 &#956;M bFGF solution (a ligand for FGFR2) to the dish and rocking gently for 60 min at room temperature.</li>
	<li>Repeat steps 3.1-3.7. 
	NOTE: Reduce the coating concentration of the FGFR2 protein to 2.5 &#956;g/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.</li>
</ol>
7. Acquisition of plaque DNA for sequencing
<ol>
	<li>Plaque amplification
	<ol>
		<li>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.</li>
		<li>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 &#60; 1&#8211;3 days old, stored at 4 &#176;C, and have &#60; 100 plaques.</li>
		<li>Incubate the tubes with vigorous shaking for 4.5 h at 37 &#176;C.</li>
		<li>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.</li>
		<li>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 &#176;C for later use.</li>
	</ol>
	</li>
	<li>Extraction of plaque DNA
	<ol>
		<li>Transfer 500 &#181;L 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&#8211;20 min at room temperature.</li>
		<li>Centrifuge at 12,000 x g for 10 min at 4 &#176;C and then pour off the supernatant. 
		NOTE: Phage pellet may not be visible.</li>
		<li>Centrifuge again. Carefully remove any remaining supernatant.</li>
		<li>Resuspend the pellet thoroughly in 100 &#181;L of the iodide buffer by vigorously tapping the tube.</li>
		<li>Add 250 &#181;L of 100% ethanol and incubate for 15 min at room temperature.</li>
		<li>Centrifuge at 12,000 x g for 10 min at 4 &#176;C and remove the supernatant. Wash the pellet with 0.5 mL of 70% ethanol, quickly centrifuge again, remove the supernatant, and brie&#64258;y dry the pellet.</li>
		<li>Resuspend the pellet in 30 &#181;L of TE buffer and use this for sequencing. 
		NOTE: Pre-cool the 70% ethanol at -20&#176;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.</li>
	</ol>
	</li>
</ol>
8. Identification of the peptide sequence
<ol>
	<li>Use the -96 gIII sequencing primer (5&#180;-CCC TCA TAG TTA GCG TAA CG &#8211;3&#180;) for sequencing.</li>
	<li>Analyze the peptide sequences displayed on the phage based on the DNA sequencing results.</li>
	<li>Synthesize small peptides and analyze by high-performance liquid chromatography (HPLC) and mass spectrometry to confirm their purity (&#8805;98%).</li>
</ol>
9. Detecting the affinity of the obtained small peptide and extracellular protein of FGFR2 by ITC
<ol>
	<li>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.</li>
	<li>Perform the ITC titration experiments at 25 &#176;C. Titrate 40 &#956;M of small peptide in the cell with 1.5 &#956;M of FGFR2 protein from the syringe, with a stirring speed of 750 rpm. Add the FGFR2 protein in 2 &#956;L aliquots (for a total of 19 injections) at 5-min intervals.</li>
	<li>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 &#176;C.</li>
</ol>
10. Verifying the biological activity of the obtained small peptide using a cell proliferation assay
<ol>
	<li>Seed BALB/c 3T3 cells (3.0 &#215; 103 cells/well, 100 &#956;L) in a 96-well plate with Dulbecco&#39;s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) overnight at 37 &#176;C and 5% CO2.</li>
	<li>Replace the supernatant with fresh DMEM with 0.5% FBS and starve the cells for 12 h.</li>
	<li>Make serial dilutions of the small peptide (0, 0.0064, 0.032, 0.16, 0.8, 4, 20, and 100 &#956;M) with fresh DMEM supplemented with 0.5% FBS (6 duplicate wells /concentration). Remove the supernatant from the 96-well plate and add 100 &#956;L of the small peptide solution per well. Incubate for 48 h at 37 &#176;C and 5% CO2.</li>
	<li>Replace the supernatant with fresh DMEM containing 10% Cell Counting Kit (CCK)-8 solution and incubate for 2 h.</li>
	<li>Measure the absorbance of each well at 450 nm with a microplate reader and analyze cell viability.</li>
</ol>

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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. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+vasculature+address+book:+Identification+of+stage-specific+tumor+vessel+zip+codes+by+phage+display.">Tumor vasculature address book: Identification of stage-specific tumor vessel zip codes by phage display.</a> Cancer Cell. 4, 331-333 (2003).</li><li>Arap, W., 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=Cancer+Treatment+by+Targeted+Drug+Delivery+to+Tumor+Vasculature+in+a+Mouse+Model.">Cancer Treatment by Targeted Drug Delivery to Tumor Vasculature in a Mouse Model.</a> Science. 279, 377-390 (1997).</li></ol>

The authors declare no conflict of financial interests.

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 ...

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

Research

JoVE Journal

Bioengineering

7.5K Views.  Guangdong Provincial Key Laboratory of Bioengineering Medicine. 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.

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

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr&#160;period.

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the efficiency with which macromolecules penetrate the cytosolic space of cells. Recently, we have circumvented this problem by identifying the reagent dfTAT, a disulfide bond dimer of the peptide TAT labeled with the fluorophore tetramethylrhodamine. We have generated a fluorescently labeled dimer of the prototypical cell-penetrating peptide (CPP) TAT, dfTAT, which penetrates live cells and reaches the cytosolic space of cells with a particularly high efficiency. Cytosolic delivery of dfTAT is achieved in multiple cell lines, including primary cells. Moreover, delivery does not noticeably impact cell viability, proliferation or gene expression. dfTAT can deliver small molecules, peptides, antibodies, biologically active enzymes and a transcription factor. In this report, we describe the protocols involved in dfTAT synthesis and cellular delivery. The manuscript describes how to control the amount of protein delivered to the cytosolic space of cells by varying the amount of protein administered extracellularly. Finally, the current limitations of this new technology and steps involved in validating delivery are discussed. The described protocols should be extremely useful for cell-based assays as well as for the ex vivo manipulation and reprogramming of cells.

We describe how to deliver proteins and cell-impermeable small molecules into cultured mammalian cells by a simple co-incubation protocol with a reagent that causes endocytic organelles to become leaky.

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used in cancer treatment and imaging due to increased cellular accumulation over current ACs. During early AC in vitro development, fluorescence techniques and radioimmunoassays are sufficient for determining intracellular localization, accumulation efficiency, and target cell specificity. Currently, there is no consensus on standardized methods for preparing cells for evaluating AC intracellular accumulation and localization. The initial testing of ACs modified with virus-derived peptides is critical especially if several candidates have been constructed. Determining intracellular accumulation by fluorescence can be affected by background signal from ACs at the cell surface and complicate the interpretation of accumulation. For radioimmunoassays, typically treated cells are fractionated and the radioactivity in different cell compartments measured. However, cell lysis varies from cell to cell and often nuclear and cytoplasmic compartments are not adequately isolated. This can produce misleading data on payload delivery properties. The intravenous injection of radiolabeled virus-derived peptide-modified ACs in tumor bearing mice followed by radionuclide imaging is a powerful method for determining tumor targeting and payload delivery properties at the in vivo phase of development. However, this is a relatively recent advancement and few groups have evaluated virus-derived peptide-modified ACs in this manner. We describe the processing of treated cells to more accurately evaluate virus-derived peptide-modified AC accumulation when using confocal microscopy and radioimmunoassays. Specifically, a method for trypsinizing cells to remove cell surface bound ACs. We also provide a method for improving cellular fractionation. Lastly, this protocol provides an in vivo method using positron emission tomography (PET) for evaluating initial tumor targeting properties in tumor-bearing mice. We use the radioisotope 64Cu (t1/2 = 12.7 h) as an example payload in this protocol.

Viral-derived peptides coupled to antibody-conjugates (ACs) is an approach gaining momentum due to the potential of delivering molecular payloads with increased tumor cell accumulation. Utilizing common methods to evaluate peptide conjugation, AC and payload intracellular accumulation, and tumor targeting, this protocol helps researchers during the key initial development phases.

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

Synthesis of Monocyte-targeting Peptide Amphiphile Micelles for Imaging of Atherosclerosis

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. Atherosclerosis is also the leading cause of sudden death and myocardial infarction, instigated by unstable plaques that rupture and occlude the blood vessel without warning. Current imaging modalities cannot differentiate between stable and unstable plaques that rupture. Peptide amphiphiles micelles (PAMs) can overcome this drawback as they can be modified with a variety of targeting moieties that bind specifically to diseased tissue. Monocytes have been shown to be early markers of atherosclerosis, while large accumulation of monocytes is associated with plaques prone to rupture. Hence, nanoparticles that can target monocytes can be used to discriminate different stages of atherosclerosis. To that end, here, we describe a protocol for the preparation of monocyte-targeting PAMs (monocyte chemoattractant protein-1 (MCP-1) PAMs). MCP-1 PAMs are self-assembled through synthesis under mild conditions to form nanoparticles of 15 nm in diameter with near neutral surface charge. In vitro, PAMs were found to be biocompatible and had a high binding affinity for monocytes. The methods described herein show promise for a wide range of applications in atherosclerosis as well as other inflammatory diseases.

This paper presents a method involving the synthesis and characterization of monocyte-targeting peptide amphiphile micelles and the corresponding assays to test for biocompatibility and ability of the micelle to bind to monocytes.