Using Phage Display to Select Proteins with High Affinity to a Target Protein

Abstract

Source: Zhang, W., et al., Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases. J. Vis. Exp. (2021)

In this video, we demonstrate the phage display technique to isolate pre-engineered phages expressing proteins with high affinity to the target protein.

Protocol

1. Prepare the phage library

1. Thaw the phage library on ice and dilute it to 100x the library diversity in PBS. For example, if the library diversity is 1 x 10¹⁰ and the library concentration is 1 x 10¹³, dilute the library so that the concentration is 1 x 10¹². For the libraries used here, dilute them 10-fold in PBS by combining 1 mL of the library with 9 mL of PBS.
   NOTE: Library diversity should have been determined during library creation and cannot be easily assessed otherwise. Library concentration can be determined by inoculating E. coli cells in the mid-log phase (OD600 0.6 - 0.8) and plating on LB/carb.
2. Add 1/5 volume of PEG/NaCl. For example, for 10 mL of the previously diluted library, add 2 mL of PEG/NaCl.
3. Incubate on ice for 30 min.
4. Centrifuge at 11,000 x g for 30 min at 4 °C, discard supernatant, and re-centrifuge for 2 min to pull down the remaining supernatant.
   NOTE: Put the tube in the rotor in the same orientation the second time as it was the first to keep the pellet in the same place, therefore, making it easier to see.
5. Gently resuspend the phage pellet in 1 mL of PBT per protein. For four proteins, resuspend the pellet in 4 mL of PBT.
   NOTE: Try not to touch the pellet and do not introduce air bubbles.

2. Display phage to the target protein(s)

1. Optional control: Add 100 µL of phage library to each coated well in the control plate. Incubate at room temperature (~20-25 °C) for 1 h with 300 rpm orbital shaking and transfer the library from the control plate to the target plate.
2. Remove the PB buffer from the target plate and add 100 µL of the phage library to each coated well.
3. Incubate at room temperature (~20-25 °C) for 1 h with 300 rpm orbital shaking.

3. Elute round one phage

1. Remove the phage library and wash the coated wells four times with PT buffer. Invert the plate and tap on a paper towel to remove the last drops.
   NOTE: If multiple target proteins are coated on one plate, try to minimize the flow of all subsequent solutions between the wells.
2. Add 100 µL of 0.1 M HCl to each coated well and incubate at room temperature for 5 min with 300 rpm orbital shaking.
3. Neutralize the pH by adding 12.5 µL of 1 M Tris-HCl (pH 11) to each coated well.
4. Pool the eluted phage from all 8 wells into a single 1.5 mL microcentrifuge tube. Pipette up and down during the transfer to make solutions homogenous and aspirate all liquid from the wells.
5. Add 10% BSA to the pooled eluted phage to a final concentration of 1%. For a typical round one elution volume of 950 µL, add 95 µL of 10% BSA. Store at 4 °C. This is the round one output.

4. Preparation for subsequent rounds of selection

1. Prepare a seed culture for culturing the phage input for the next round of selection as in step 1.1.
2. Coat the plate for the third round of selection as in step 1.2 with the changes noted below.
   1. Only coat four wells per protein in a 96-well binding plate. Use half of the contents of the "round 2/3" or "round 4/5" tubes for the appropriate round. Dilute the contents of these tubes as needed, in order to avoid proteins settling out of the solution.
3. Prepare the phage input for the next round of selection.
   1. Use half of the round one output to inoculate 3 mL of mid-log phase cells. For a typical round one output, inoculate with 500 µL of the output.
   2. Incubate at 37 °C for 30 min with 200 rpm orbital shaking.
   3. Add M13K07 helper phage to a final concentration of 1 x 10¹⁰ PFU/mL.
   4. Incubate at 37 °C for 1 h with 200 rpm orbital shaking.
   5. Transfer the entire 3 mL of culture to 30 mL of 2YT/carb/kan. Grow overnight at 37 °C with 200 rpm orbital shaking.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Axygen Mini Tube System (0.65 mL, sterile, 96/Rack, 10 Racks/pack)	Fisher Scientific	14-222-198	Plastic ware
BD Difco Dehydrated Culture Media: LB Broth, Miller (Luria-Bertani)	Fisher Scientific	DF0446-17-3	Preparing plates for titering
Bovine Serum Albumin (BSA), Fraction V	BioShop Canada	ALB001	Buffer component
Carbenicillin disodium salt 89.0-100.5% anhydrous	Millipore-Sigma	C1389-5G	Culturing phagemid-infected cells
Compact Digital Microplate Shaker	Fisher Scientific	11-676-337	Device
Corning Microplate Aluminum Sealing Tape	Fisher Scientific	07-200-684	Sealing phage glycerol stocks
Dehydrated Agar	Fisher Scientific	DF0140-01-0	Preparing plates for titering
DS-11 Spectrophotometer/Fluorometer	DeNovix	DS-11 FX+	Device
Greiner Bio-One CellStar 96-Well, Non-Treated, U-Shaped-Bottom Microplate	Fisher Scientific	7000133	Plastic ware
Hydrochloric Acid	Fisher Scientific	A144-500	Chemical
Invitrogen One Shot OmniMAX 2 T1R Chemically Competent <em>E. coli</em>	Fisher Scientific	C854003	Bacterial strain for phage infection
Kanamycin Sulfate	Fisher Scientific	AAJ1792406	Culturing M13K07 helper phage-infected cells
M13KO7 Helper Phage	New England Biolabs	N0315S	Permit phagemid packing and secretion
MaxQ 4000 Benchtop Orbital Shaker	Fisher Scientific	11-676-076	Device
Nunc MaxiSorp 96-well microplate, flat bottom	Life Technologies	44-2404-21	Plastic ware
Phosphate-Buffered Saline (PBS) 10X Solution	Fisher Scientific	BP3994	Buffer component/phage resuspension medium
Polyester Films for ELISA and Incubation	VWR	60941-120	Covering the microplates during incubation
Polyethylene Glycol 8000 (PEG)	Fisher Scientific	BP233-1	Phage precipitation
Sodium chloride	Millipore-Sigma	S3014	Phage precipitation
Sterile Plastic Culture Tubes: Translucent Polypropylene	Fisher Scientific	14-956-1D	Culturing phage inputs
Tetracycline Hydrochloride	Fisher Scientific	BP912-100	Culturing <em>E. coli</em> OmniMax cells
Tris Base	Fisher Scientific	BP1525	Neutralizing eluted phage solution
Tryptone Powder	Fisher Scientific	BP1421-2	Cell growth media component
Tween 20	Fisher Scientific	BP337500	Buffer component
Yeast Extract	Fisher Scientific	BP1422-2	Cell growth media component