E coli er2738 host strain

The Ph.D.-7 Phage Display Library Kit including the Heptapeptide Phage Display Library and E. coli ER2738 host strain were purchased from New England Biolabs. ER2738 with F factor, which confers tetracycline resistance, can grow in LB-Tet medium (1% bacto-tryptone, 0.5% yeast extract, 0.5% NaCl, 20 g/mL tetracycline) and is used for M13 phage propagation. Since the library phage carries the lac Z gene, phage plaques appear blue when the phage-infected ER2738 cells are plated on LB-Tet-IPTG-X-gal media (1% bacto-tryptone, 0.5% yeast extract, 0.5% NaCl, 20 µg/mL Tetracycline, 50 µg/mL IPTG, 40 µg/ml X-gal, 1% agarose).

Target proteins (folate receptor-α, programmed death 1 and programmed death-ligand 1) were dissolved in carbonate buffer pH 9.6 and coated on polystyrene 96-well microtiter plates (Corning, Inc., NY, USA) overnight at 4 °C at a concentration of 50–150 μg/mL. The next day, each well was blocked with 250 μL 3% (w/v) non-fat milk (NFM) (Sangon Biotech Co., Ltd., Shanghai) for 2 hours at 37 °C. After washing 5 times with TBS containing 0.05% (v/v) Tween 20 (TBST), 100 μL Ph.D.-12 peptide library (New England BioLabs, Inc., USA) aliquot containing 10¹¹ phages was added to each well and the plate was incubated for 1 hour at 37 °C. After washing 10 times with TBST and 3 times with TBS to remove the unbounded phages, the target-bound phages were eluted with 150 μL 100 mM Glycine-HCl (pH 2.2) and neutralized immediately with 9 μL 2 M Tris-HCl (pH 9.1). 5 μL of eluate was used for titering and the rest was used to infect E. coli ER2738 host strain (New England BioLabs, Inc., USA) for amplification.