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

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