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(beta-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 to have adenylate cyclase enzymatic activity which was activated by calmodulin, to bind 125I-calmodulin, and to be free of pertussis toxin as determined by in vivo and in vitro assays.