Ubiquitination is a critical protein post-translational modification, dysregulation of which has been implicated in numerous human diseases. This protocol details how phage display can be utilized to isolate novel ubiquitin variants that can bind and modulate the activity of E3 ligases that control the specificity, efficiency, and patterns of ubiquitination.
Phage display not only helps us investigate the binding characteristics of clinically important enzymes, but also develop modulators for those enzymes and the corresponding diseases. Phage display can be used to develop novel binders for a wide array of target proteins and is also easier to perform than other conventional display methods. This procedure involved a lot of repetitive action, so the key is to not autopilot and to remain focused throughout.
Begin by inoculating 30 milliliters of 2YT tetracycline broth with 200 microliters of the seed culture. Incubate it for approximately three hours at 37 degrees Celsius with 200 RPM orbital shaking until the bacteria are in the mid-log phase. Discard the coating solution from the plate into a sink.
Dry it gently with paper towels. Then add 200 microliters of PB buffer to each coated well. Discard the PB buffer from the plate and dry it on paper towels.
Add another 200 microliters of PB buffer to each coated well of the plate. Incubate it at room temperature for an hour with 300 RPM orbital shaking. Thaw the phage library on ice, then dilute it to 100X the library diversity in PBS.
Add the polyethylene glycol sodium chloride solution at one-fifth of the diluted library volume and incubate the solution on ice for 30 minutes. Next, centrifuge the solution at 11, 000 times G for 30 minutes at four degrees Celsius. Discard the supernatant and centrifuge it for another two minutes to pull down the remaining supernatant and concentrate the phage pellet.
Gently resuspend the phage pellet in one milliliter of PBT buffer per target protein to be analyzed, typically four in total. Add 100 microliters of phage library to each coated well in the control plate. Incubate it at room temperature for an hour with 300 RPM orbital shaking.
Discard the PB buffer from the target plate into the sink and dry the plate on paper towels. Transfer all 100 microliters of the phage library in the control plate to each coated well of the target plate. Incubate it at room temperature for an hour with 300 RPM orbital shaking.
Next, remove the phage library from the plate and wash the coated wells four times with PT buffer. Invert the plate and tap on a paper towel to remove the last drops of buffer. Add 100 microliters of 0.1 molar hydrochloric acid to each coated well to elute the phage.
Incubate the plate at room temperature for five minutes with 300 RPM orbital shaking. After that, neutralize the pH by adding 12.5 microliters of pH 11 one molar tris hydrochloride to each coated well. Transfer the eluted phage from all eight wells into a single 1.5 milliliter microcentrifuge tube.
Pipette up and down during the transfer to make solutions homogenous and aspirate all liquid from the wells. Add 10%BSA to the eluted phage to make the final concentration 1%Store the microcentrifuge tube at four degrees Celsius. This is the round one output.
Prepare a seed culture for culturing the phage input for the next round of selection by inoculating five milliliters of 2YT tetracycline seed culture with an isolated E.coli colony from an agar plate. Incubate it overnight at 37 degrees Celsius with 200 RPM orbital shaking. Dilute the target protein in the rounds two and three tube with an appropriate amount of PBS.
Take half of the contents of rounds two and three tube and coat four wells per target protein in a 96-well binding plate for the next round. Next, use half of the round one output to inoculate three milliliters of mid-log phase cells. Incubate it at 37 degree Celsius for 30 minutes with 200 RPM orbital shaking.
Add M13KO7 helper phage to a final concentration of 10 billion PFU per milliliter. Incubate it again at 37 degrees Celsius for an hour with 200 RPM orbital shaking. After an hour, transfer the entire three milliliters of culture to 30 milliliters of 2YT carbenicillin kanamycin solution.
Grow overnight at 37 degrees Celsius with 200 RPM orbital shaking. The representative results for a ubiquitin variant selection against UBE4B are shown here. UBVs were ordered from the highest to lowest frequency.
Sequences represent the diversified ubiquitin region in the UBV with all the randomized residues. The relative binding affinity of the binders to the wild type target protein, mutated target protein, as well as off-target proteins is measured by enzyme-linked immunosorbent assays or ELISA. All ELISA absorbances were normalized against 96 averaged BSA ELISA scores and 96 averaged GST ELISA scores.
Darker green represents the stronger relative binding. GST was included as a control for nonspecific binding because the target protein is GST tagged. The ELISA results are graphically presented here.
The x-axis represents the UBVs and the y-axis represents the corresponding normalized absorbance. When attempting this procedure, it's important that you don't discard the solution in the plate after you add the hydrochloric acid. The phages are now suspended in solution and you want to keep these so that you can perform subsequent enrichment final analyses or both.
Following this procedure, sequencing should be done to determine UBV frequencies. ELISAs and IC50 assays can be done to determine binding efficacies and inhibitory efficacies respectively and also to help determine which UBVs to further characterize.
