Figure 5
Panel A. Western blot analysis of the bacterial lysates (BL), periplasmic extracts (PE) and soluble total extracts (TE) obtained from equal volumes of bacterial cultures expressing the scFv 43 M1 and M2 mutants, and scFv43. The Western blotting was performed using the anti-FLAG M2 mAb as a primary antibody, followed by GAM-HRP IgG incubation. The immunocomplexes were revealed by chemiluminescence. The molecular mass markers are indicated on the left. The scFv molecular weight of about 27.5 KDa is indicated by an arrow. Panel B. Analysis of the scFv 43 M1 and scFv43 M2 thermal stability in comparison with that of the parental scFv43. The scFvs were incubated at 37°C for different time intervals, indicated on the abscissa. Note that the scale is not linear. At the end of the incubation the residual reactivity towards the recombinant E7 protein was evaluated by ELISA; the OD values at 450 nm are reported on the ordinate. The t1/2, defined as the time of halving of the anti-E7 reactivity after incubation at 37°C, is indicated by an arrow for each scFv.