Objective: Cryptosporidiosis, caused by Cryptosporidium species, is a common zoonotic disease all over the world. Cryptopain, an important virulence factor of Cryptosporidium spp., is a cysteine protease inhibitor, and cryptostatin is a cysteine protease inhibitor. Cysteine proteases are virulence factors that play an important role in many parasites and are therefore among the targets for drug designs.

Methods: Modified Kinyoun acid-fast staining was performed using the feces of animals that may harbor Cryptosporidium spp., and DNA isolation was performed from the positive samples. Cryptopain-1 and cryptostatin genes were cloned. After cloning, total protein extract was prepared and purified. Purified proteins were evaluated by SDS-Page imaging. Gelatinase activity of purely obtained cryptopain and cryptostatin were tested on medium containing three percent gelatin. Moreover, the activities of cryptopain and cryptostatin at different pH and temperature were tested.

Results: Our results showed that recombinant cryptopain, a cysteine protease, had gelatinase activity, however in presence of recombinant cryptostatin and control group, no gelatinase activity was observed. In the IgG and BSA degradation evaluations of the purified proteins, it was determined that cryptopain-1 degraded both proteins at different temperatures and pH values. Cryptostatin inhibited cryptopain activity at different temperatures and pH values.

Conclusion: Recombinant cryptostatin has potential for the inhibition of Cryptosporidium parvum proteases and prevention of infections.