A STABLE OLIGOMER OF BACILLUS-THURINGIENSIS DELTA-ENDOTOXIN, CRYIIIA

A purified sample of crystalline delta-endotoxin, CryIIIA, formed a stable oligomer in various gel systems and in solution. Non-heated samples of alkaline-solubilized CryIIIA demonstrated a high MW band with Laemmli buffer/SDS-PAGE. Furthermore, CryIIIA produced a single band with Native-PAGE migrating between BSA dimer (132,000 Da) and beta-amylase (200,000 Da) standards. A related CryIII toxin, CryIIIB2, demonstrated a similar size band on the same Native-PAGE system, suggesting a general phenomenon of CryIII oligomerization. When I-125-CryIIIA was solubilized in 0.050 M potassium phosphate, pH 7.0, and run as a non-heated sample in neutral potassium phosphate buffer SDS-PAGE, a high MW band was detected, indicating that the oligomer could theoretically form under less alkaline conditions such as would be encountered in a coleopteran midgut. Analytical ultracentrifugation of alkaline-solubilized CryIIIA provided strong evidence for a stable species in solution with a MW estimate of 117,200, 113,300 or 101,500+/-4800 for three separate sedimentation equilibrium experiments. These data demonstrate that CryIIIA most likely exists as a dimer in solution and support the premise that delta-endotoxins form oligomeric structures which may be crucial for sustaining large conductance ion channels in host brush border membranes.