A New Functional Site W115 in CdtA Is Critical for Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin
Figure 4
Effect of W115G mutation in CdtA on the holotoxin assembly.
The ability of mutated CdtAW115G subunit to form a stable heterotoxin complex was determined by size exclusion chromatography. The CDT holotoxin was reconstituted by co-refolding all three subunits together via dialysis at 4°C into a native buffer consisting of 10 mM Tris and 75 mM NaCl (pH 7.4). A total of 1.5 ml protein mixtures were injected onto a HiLoad 16/60 Superdex 200 prep grade column and run at flow rate of 2 ml/min in 10 mM Tris and 75 mM NaCl (pH∶7.4) on an AKTA FPLC. Fractions of 2 ml were collected. Peak fractions near 7 ml were collected and analyzed by SDS-PAGE, and the mutant CDT subunits were all visualized with Coomassie blue stain. M. molecular weight markers, A*. CdtAW115G, B. CdtB, C. CdtC.