Role of of Pro region of El Tor cytolysin

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Purified protoxin purified from E.coli harboring hlyA.The protoxin was processed to mature hemolysin by HA/protease of V.cholerae and trypsin, resulting increase in hemolytic activity. The region of hlyA coding for Pro region of was deleted and the deleted gene (hlyA-DELTA pro) cloned to a suicide vector plasmid. The plasmid was introduced to the parent strain and the native gene was replaced with hlyA-DELTA pro (N86-hlyA-DELTA pro). The jmutant strain decreased in hemolytic activity. The mutant strain did not produce mature hemolysin in the supernatant. Western blot showed that destruction of protoxin in the periplasm. Protoxin and mature toxin was denatured with guanidine-HC1 and reconstituted by decreasin the concentration of the solution. Protoxin recovered 100% hemolytic activity but mature toxin did not recover the activity. These data suggest that Pro region may play role in protective effect against proteases and renaturation of hemolsin.