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The gene encoding perfringolysin O, the thiol-activated hemolysin from Clostridium perfringens (ATCC 13124), was cloned and expressed in Escherichia coli. A gene library of C. perfringens chromosomal DNA was constructed in bacteriophage lambda EMBL3. A recombinant was identified that produced a hemolysin that was inhibited by cholesterol and was tentatively identified as perfringolysin O. Subcloning experiments localized the perfringolysin O gene (pfo) to a 1.8-kilobase region on the cloned chromosomal fragment. E. coli which carried a plasmid subclone of pfo (pRT1B) expressed perfringolysin O and secreted it into the periplasm. The amino-terminal sequence of the pfo gene product was identical with that determined for perfringolysin O purified from C. perfringens, indicating that E. coli correctly removed the signal peptide during secretion. Purification of the pfo product was accomplished by high-resolution gel filtration and anion-exchange chromatography. Analysis of the pfo product by sodium dodecyl sulfate gel electrophoresis showed that it comigrated with authentic perfringolysin O; both had an estimated molecular weight of 54,000. Two-dimensional tryptic peptide maps of the pfo product and of authentic perfringolysin O purified from C. perfringens were identical. The hemolytic activity of the pfo product was similar to that of authentic perfringolysin O; one hemolytic unit (HU) of the cloned gene product or authentic perfringolysin O corresponded to approximately 1 ng or a hemolytic activity of 10(6) HU per mg.