Figure 8. Aerolysin oligomerization is controlled by the CTP.

A: Proaerolysin was activated with trypsin agarose beads at 4°C in a Tris pH 8 buffer to inhibit oligomerization and the sample was diluted with 50 mM Hepes pH 7 to a concentration of 10 µM in order to favor oligomerization in the presence or absence of a 5-fold molar excess (i.e. 50 µM) of synthetic CTP or control peptide. The sample was incubated at room temperature for different times and subsequently analyzed by SDS-PAGE followed by Coomassie blue staining. B: The amount of oligomer was quantified for 3 independent experiments using ImageJ (n = 3). Error bars represent standard deviations. C: Proaerolysin at a concentration of 1 mg/mL in a buffer at pH 8 (to inhibit oligomerization) was activated with trypsin agarose beads and subsequently dialyzed against 10 mM Hepes buffer pH 7.4, 10 mM NaCl to initiate the oligomerization process. Two different dialysis molecular weight cut offs where used: 14 kDa, which allows the passage of the CTPs and 3.5 kDa, which retains peptides the size of the CTP. After 2 hours of dialysis at RT, the samples were analyzed by SDS-PAGE followed by Coomassie blue staining.