Cloning and expression of phosphoenolpyruvate carboxykinase from a cestode parasite and its solubilization from inclusion bodies using l-arginine

• The ORF of PEPCK from a parasite was cloned into pGEX-4T3, which expressed as IBs.
• Addition of 2 M l-arginine solubilized IBs, and rePEPCK was purified.
• A yield of ∼73 mg of purified rePEPCK was obtained per 1 L of culture.
• The specific activity and Kmapp were 21 U/mg of protein and 43 μM, respectively.

Phosphoenolpyruvate carboxykinase is an essential regulatory enzyme of glycolysis in the cestode parasite, Raillietina echinobothrida, and is considered a potential target for anthelmintic action because of its differential activity from that of its avian host. However, due to the unavailability of its structure, the mechanism of regulation of PEPCK from R. echinobothrida (rePEPCK) and its interaction with possible modulators remain unclear. Hence, in this study, the rePEPCK gene was cloned into pGEX-4T-3 and overexpressed for its characterization. On being induced by IPTG, the recombinant rePEPCK was expressed as inclusion bodies (IBs); hence, various agents, like different inducer concentrations, temperature, time, host cell types, culture media, pH, and additives, were used to bring the protein to soluble form. Finally, a significant amount (∼46%) of rePEPCK was solubilized from IBs by adding 2 M l-arginine. Near-UV circular dichroism spectra analysis indicated that l-arginine (2 M) had no effect on the conformation of the protein. In this study, we have reported a yield of ∼73 mg of purified rePEPCK per 1 L of culture. The purified rePEPCK retained its biological activity, and Km of the enzyme for its substrate was determined and discussed. The availability of recombinant rePEPCK may help in biochemical- and biophysical-studies to explore its molecular mechanisms and regulations.