FIG 2.

Trypsin sensitivity of natural CPB variants. (A) Overnight cell-free TGY culture supernatants were produced for strains CN3685 and JGS1076 (representing each CPB variant) and subjected to digestion with various doses of trypsin at 37°C for 30 min. SDS-PAGE and Western blotting were performed to detect undigested CPB. A representative blot of CPB remaining after digestion is shown for each trypsin dose. Each digestion was performed twice. (B and C) An overnight TGY culture of a CN3685 CPB null mutant was used to generate a cell-free culture supernatant. Purified CPB (40 μg/ml) from both variants was used to spike culture supernatant (B) or HBSS (C), followed by digestion with trypsin. Western blotting and densitometry were performed to determine the amounts of CPB remaining relative to starting amounts of the toxin. The times for 50% digestion from four replicate digests are presented. Error bars indicate standard errors of the means. #, there was a significant (P < 0.05) difference in digestion times.