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Isolation of the complex between rBbCI, a recombinant Bauhinia bauhinioides Cruzain Inhibitor, and the major cysteine proteinase from T. cruzi
Hansen, D. 1; Araújo, A. P. U. 2; Veríssimo, P. 3; Ribeiro, S. M. 3; Cezari, M. H. S. 4; Yoo Im, S. 1; Sampaio, M. U. 1 and Oliva, M. L. V. 1
1 Departamento de Bioquímica - UNIFESP - Escola Paulista de Medicina, São Paulo - SP; 2 Instituto de Física de São Carlos - USP, São Carlos - SP; 3 Centro de Neurociências e Biologia Celular - Universidade de Coimbra, Portugal; 4 Departamento de Biofísica - UNIFESP - Escola Paulista de Medicina, São Paulo - SP.
Infection by the protozoan parasite Trypanosoma cruzi results in Chagas' disease, which is the principal cause of heart disease in South America. The only currently available treatment with Nifurtimox e Benzonidazol is insufficient due to considerable toxicity. The lack of effective treatment has stimulated efforts to identify novel drugs against the parasite. One target that has received significant attention is cruzain, the major cysteine proteinase found in T. cruzi. Studies shown that irreversible inhibitors of cruzain can cure parasitic infections in mouse models.
In our laboratory, a plant Kunitz type cruzain reversible inhibitor, BbCI, isolated from Bauhinia bauhinioides seeds, displays promising features for the treatment of Chagas' disease.
Our work has the aim to study the complex cruzain and BbCI, mainly its interactions in the inhibitor reactive site. For this purpose, we produced both the recombinant molecules, BbCI as previously described, and the cruzain, obtained by heterologous expression in E. coli, denaturation and refolding, as described by Eakin et al., 1992.
The complex isolation was performed by incubation at 2:1 ratio of rBbCI - cruzain, in ice bath for 2 hours, followed by gel filtration purification in Superdex 75 column. The fraction corresponding to the complex was concentrated in Centriprep 10 kDa and submitted to crystallization screening. The optimal condition was that of hanging drop at 18 oC, in 1M sodium citrate pH 7.0.
Supported by FAPESP, CNPq and CAPES-GRICES-DAAD.
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