Oxidative Folding of Cruzain, the Cysteine Protease Involved in the Progression of Chagas Disease

Chagas Disease is endemic to Latin American and is gradually making its way to other countries.  The etiological agent of the disease is Trypanosoma cruzi, a protozoan parasite that causes lesions and inflammation of vital organs.  No vaccine is available and current treatment is effective only during early onset.  One target for treatment is inhibiting cruzain, the cysteine protease believed to be important to the progression of the disease.   Current research is being conducted on inhibiting the active site of cruzain however, the safety of inhibitors as treatment is unknown.  In our study, we would like to prevent the protein from gaining three dimensional structure and prevent its function.  We are studying the folding pattern of the protein to identify stable intermediates that can be targeted by small molecule inhibitors.  We believe that cruzain will fold and regain its activity and that the rates of folding of the active enzyme and the inactive proenzyme will differ.  Here, we describe a detailed method for expression, purification, activation of cruzain as well as oxidative folding of the enzyme.  It has have proven that the active protein will refold and regain its activity and that the proprotein has a slower folding rate than the mature enzyme.  This study gives an introduction to a back door approach in treating Chagas Disease and will lead to the identification of folding intermediates of procruzain and cruzain.