Cloning and recombinant expression of Caspase-activated DNase orthologous gene of Giardia lamblia

In eukaryotic cells, mitochondria play a key role in apoptosis, but Giardia lamblia only possesses a mitochondrial remnant, the mitosome. However, this protozoan can die by an apoptosis-like process, probably by an apoptosis-mitochondrial-independent pathway. Here, we aimed to identify, clone, express, and characterize the caspase-activated DNase (CAD) of G. lamblia . Using a commercial polyclonal antibody that recognizes mouse, rat, and human CAD (hCAD), we developed an immunoproteomic analysis using a crude extract of curcumin-treated G. lamblia trophozoites (CEGl), detected a spot of 42kDa, and pI 9.4, similar to hCAD. This spot was sequenced by LC-MS. The proteomic profile (eleven peptides) matched with a novel protein of 383 residues, with a predicted 42KDa, pI 9.4, with a CIDE-N domain and putative H-K-H cata-lytic motif. Afterward, we cloned the full-length gene (GenBank: ON707040), expressed it, and purified it as a H 6 tag-recombinant protein in Escherichia coli , which also was recognized by commercial anti-CAD. In conclusion, gCAD is an orthologous protein of hCAD and will allow us to determine its role in the apoptosis-like signaling pathway in G. lamblia .