Cloning and Recombinant Expression of the Caspase-Activated DNase Orthologous Gene of Giardia lamblia.

Abstract

In eukaryotic cells, mitochondria play a key role in apoptosis; however, ancestral eukaryotic cells such as Giardia lamblia only possess a mitochondrial remnant, the mitosome. Interestingly, this protozoan still undergoes an apoptosis-like process; therefore, we focused primarily on the search for the mitochondria-independent executor DNase. Here, we identified, cloned, expressed, and characterized the caspase-activated DNase (CAD) from Giardia lamblia. Using a commercial polyclonal antibody that recognizes mouse, rat, and human caspase-activated DNase (hCAD), we developed an immunoproteomic analysis using a crude extract of curcumin-treated Giardia lamblia trophozoites (CEGl) and detected a spot of 42 kDa and pI 9.4, similar to hCAD and sequenced by LC-MS. The proteomic profile matched a novel protein of 383 residues, with a predicted 42 kDa, pI 9.4, a CIDE-N domain, and putative H-K-H catalytic motif. Afterward, we cloned the full-length gene (GenBank: ON707040), expressed it, and purified it as a 6-His tag-recombinant protein in Escherichia coli, which was also recognized by commercial anti-CAD. In conclusion, genetic, proteomic, and structural analyses showed that the identified gCAD is an orthologous protein of hCAD, and its DNase role in the apoptosis-like signaling pathway of Giardia lamblia can be further analyzed.