Cloning Dictyostelium Paracaspase Protein in the Acanthamoeba Expression Plasmid

Abstract

Acanthamoeba castellanii and the slime mold Dictyostelium discoideum are members of Amoebozoa. Both microorganisms were applied as model organisms in different biological studies. A. castellanii metacaspase (Acmcp) and D. discoideum paracaspase (Ddpcp) proteins have been recently discovered. Determining the function of Acmcp and Ddpcp provides a valuable information about their role in more complex organisms. This study aims to clone the Ddpcp gene in the Acanthamoeba expression plasmids. The Ddpcp gene was inserted  in a plasmids containing the TBP Promoter Binding Factor (TPBF) gene promoters from A. castellanii and enhanced green fluorescent protein (EGFP) as the reporter gene. The promoters for Acanthamoeba TPBF gene was used to drive constitutive expression of EGFP protein in stably transfected Acanthamoeba. Recombinant TPBF is able to bind DNA and activate transcription as the natural Acanthamoeba TPBF. The results showed a sucssesful constract of  production of recombinant Ddpcp gene in pTPBf plasmid that will be able to use later to provide a valuable information about the function of Ddpcp in the Acanthamoeba parasite.