Construction of the Libraries of LC3B and GATE-16 Variants, Selections of the LC3B/GATE-16 Variants and Their Characterization by IC50 Phage ELISA

M13 phage display libraries of human LC3B and GATE-16 variants were constructed as described before using soft randomization strategy (Fellouse and Sidhu, 2007; Ernst et al., 2013; Wiechmann et al., 2017). The libraries were used for solid phase phage panning experiments and the selection stringency during the subsequent panning steps was increased by extending the number of washing cycles, shifting the incubation/washing from 4°C to room temperature, decreasing the amounts of coated target proteins and increasing the present amounts of the counter selection proteins. Individual clones were analyzed by phage colony and, subsequently, phage specificity ELISA and DNA sequencing after three to five rounds of selection. Phage IC₅₀ ELISA was performed as described previously (Wiechmann et al., 2017) but using 1 μM, 500 nM, 250 nM, and 125 nM concentrations of the free binding protein in solution.