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

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)129-134
Number of pages6
JournalInfection and Immunity
Volume55
Issue number1
DOIs
StatePublished - 1987

ASJC Scopus subject areas

  • Parasitology
  • Microbiology
  • Immunology
  • Infectious Diseases

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