Skip to main content
. 2017 Dec 12;8:1759. doi: 10.3389/fimmu.2017.01759

Figure 5.

Figure 5

Impact of stability selection on human VH/VL single-domain antibody (sdAb) library sequence diversity. After transformation of Escherichia coli TG1 cells with phagemid DNA, phage were rescued by superinfection of overnight cultures with M13K07 helper phage or M13K07ΔpIII hyper phage. The purified sdAb-displaying phage were bound and eluted from protein A (VHB82SS library) or protein L (VL383SS library), then amplified by reinfection of E. coli TG1 cells. Three rounds of panning were performed, and VH/VL sdAb sequences were interrogated by NGS at the stage of rescued phage, phage eluted after a single round of protein A/L selection, and phage amplified after the final round of panning. (A,B) Proportion of functional sdAb sequences (in-frame ORF, no stop codons) observed in VL (A) and VH (B) library phage and panning outputs. (C,D) Clonality of VL (C) and VH (D) library phage and panning outputs. (E,F) Complementarity-determining region (CDR) amino acid composition of enriched VL (E) and VH (F) sdAb clones after three rounds of stability selection. Crossed-out cells indicate a frequency of <0.1%. Analyses in (A–F) are representative of 7.7 × 104 to 7.9 × 105 sequences per sample.