Previously the 126-kDa Bordetella pertussis CyaA pore-forming (CyaA-PF) domain expressed in Escherichia coli was shown to retain its hemolytic activity Here a 100-kDa RTX (Repeat-in-ToXin) sub-cloned fragment (CyaA-RTX) containing a number of putative calcium-binding repeats was further investigated The recombinant CyaA-RTX protein although expressed as a soluble form in a protease-deficient E coli strain BL21(DE3)pLysS was found to be highly sensitive to proteolytic degradation Interestingly the addition of calcium ions in a millimolar range Into the CyaA-RTX preparation significantly prevented the degradation Moreover levels of proteolytic degradation were dependent on calcium concentrations implying an Important role for calcium-binding sites in the RTX subdomain for structural stability Homology-based modeling of the repetitive blocks in the CyaA-RTX subdomain supports that this calcium-bound protein folds into a parallel beta-roll structure with calcium ions acting as a structural stabilizing bridge (C) 2010 Elsevier Inc All rights reserved