Fig. 3. Phenotypic selection of cyclonal variants with differential antigen-binding activity.
a Representative selective spot plating of SHuffle T7 Express cells carrying a plasmid encoding spTorA-CAT-Gcn4-PP and a second plasmid encoding anti-Gcn4 cyclonal parent (GLF) or variant with CDR-H3 mutation as indicated at left. A total of 5 μl of 10-fold serial diluted cells was plated on LB-agar supplemented with 0 or 20 μg/mL chloramphenicol (Cm) as well as 0.4 % arabinose and 1 mM IPTG to induce protein expression. Spot plating results are representative of at least three biological replicates. Dashed lines indicate merged images from two different spot plates. Binding affinity was determined for GLF, GLM, ALF, and GFA (see Supplementary Fig. 1) and values for KD are indicated at right. b Survival curves for serially diluted SHuffle T7 Express cells co-expressing an anti-Gcn4 cyclonal variant along with the spTorA-CAT-Gcn4-PP reporter. Cells expressing the parental GLF cyclonal along with the non-cognate spTorA-CAT-HAG chimeric antigen served as a negative control. Overnight cultures were serially diluted in liquid LB and plated on LB-agar supplemented with Cm. Maximal cell dilution that allowed growth is plotted versus Cm concentration. Arrow in (b) indicates data depicted in image panel (a) and corresponds to 20 μg/mL Cm. Data points are the average of six biological replicates ± SD. c Antigen-binding activity of GLF, GLM, ALF and GFA cyclonals as determined by ELISA with purified GST-Gcn4-PP as immobilized antigen. Absorbance was measured at 450 nm and values were normalized to the maximum value obtained for GLF. Data are the average of three biological replicates ± SD. Statistical significance was determined by two-tailed t test with Welch’s correction (*p < 0.05; **p < 0.01, ***p < 0.001; ****p < 0.0001). Actual p values in from top-to-bottom and left-to-right: p = 0.0007, p = 0.0018, p = 0.0148, p = 0.0034, p < 0.0001, p = 0.0009. Source data are provided as a Source Data file.