Bordetella pertussis adenylate cyclase: Isolation and purification by calmodulin-sepharose 4B chromatography

Purified preparations of adenylate cyclase were obtained from crude urea extracts of Bordetella pertussis by a one-step calmodulin affinity chromatography technique. Diluted extract was loaded onto the column and washed, and adenylate cyclase was eluted with 10mM EGTA [ethylene glycol-bis(β-aminoethyl ether)-N,N,N', N'-tetraacetic acid]. A 104-fold purification was accomplished in one step. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the affinity-purified adenylate cyclase was dissociated into one major protein band with an apparent molecular weight of 60,000 and a minor band at 200,000. The affinity-purified adenylate cyclase was observed (i) to have adenylate cyclase enzymatic activity which was activated by calmodulin, (ii) to bind ¹²⁵I-calmodulin, and (iii) to be free of pertussis toxin as determined by in vivo and in vitro assays.