Characterization of API P2-P1 variants identified by sequencing of plaque-purified thrombin-selected or mock-selected phages.
After biopanning the P2–P1 phage display library for five rounds with (+) or without (−) thrombin, the API inserts from 20 plaque-purified phage were sequenced from each biopanned library. Panel A shows the observed frequency, as a percentage, for variants with the dipeptide sequences shown on the x axis. Black bars correspond to variants identified by thrombin panning, while white bars correspond to those identified by mock selection; hatched bars identify the PP sequence identified in both groups. Panel B shows API variants with the same P2–P1 dipeptide sequences as in Panel A, but transferred to plasmids (pBAD) for expression as soluble, intracellular proteins. Cultures containing plasmids expressing the API P2-P1 dipeptide identified on the x axis were lysed and the extent to which anti-API immunoreactive proteins bound to thrombin immobilized on microtiter plate wells was quantified as the optical density at 450 nm. The mean of duplicate determinations ± SD is shown. As in Panel A, black bars relate to thrombin-panned and white bars to mock-selected candidates. Cross-hatched bar shows the results from a colony transformed with pBAD-H6API M358R as a positive control.