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
