Recovery of AC enzymic activity of CyaA of Bordetella pertussis in the absence of 8 M urea with new purification methods

Adenylate cyclase toxin (CyaA) toxin is an important virulence factor of Bordetella pertussis, the causative agent of whooping cough, and a potential component of acellular pertussis vaccine. The aims of this study were to remove the urea, normally used to stabilize the protein, and to determine the stability of the enzymic and cytotoxic activities. The work was designed to produce the native enzymatically-active, invasive toxin (CyaA) as recombinant proteins. The AC enzymic activity was assayed by a conductimetric method. The toxins were purified by column chromatography using Q and Butyl Sepharose as new method. Produced CyaA was formulated as protein-coated microcrystals (PCMCs) on the surface of microcrystals of DL-valine. CyaA coprecipitated with different combination of CaM, BSA, CaCl2 or ATP and crystals were dissolved in different buffers at various pHs. The CyaA in the new formulation was shown not to be readily soluble in aqueous buffers, but could be solubilised in urea buffers and retained high AC and cytotoxic activity. Many different types of PCMC formulation were prepared in order to increase solubility of PCMCs. The most promising results were obtained when PCMCs were dissolved in 100mM Bicine (pH 8). It was clear that the combination of these two were a more suitable method for CyaA purification. The results indicated that CyaA-CaM-BSA-PCMCs offer a promising way to preserve the activity and antigenicity of CyaA in non-aqueous formulation. Such production could have application for presentations of protein antigens that normally require cold storage for stability.

Keywords: Adenylate cyclase toxin, B. pertussis, PCMCs, AC activity

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