title: Cloning and expression of phosphoenolpyruvate carboxykinase from a cestode parasite and its solubilization from inclusion bodies using l-arginine.
abstract: 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 2M l-arginine. Near-UV circular dichroism spectra analysis indicated that l-arginine (2M) had no effect on the conformation of the protein. In this study, we have reported a yield of ∼73mg of purified rePEPCK per 1L 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.
authors.affiliation: Department of Zoology, North-Eastern Hill University, Shillong, Meghalaya 793022, India.
authors.forename: Asim K
authors.initials: AK
authors.lastname: Dutta
authors.affiliation: Department of Zoology, North-Eastern Hill University, Shillong, Meghalaya 793022, India.
authors.lastname: Ramnath
authors.affiliation: Department of Zoology, North-Eastern Hill University, Shillong, Meghalaya 793022, India.
authors.forename: Barilin
authors.initials: B
authors.lastname: Dkhar
authors.affiliation: Department of Zoology, North-Eastern Hill University, Shillong, Meghalaya 793022, India.
authors.forename: Veena
authors.initials: V
authors.lastname: Tandon
authors.affiliation: Department of Zoology, North-Eastern Hill University, Shillong, Meghalaya 793022, India. Electronic address: dasbidyadhar@gmail.com.
authors.forename: Bidyadhar
authors.initials: B
authors.lastname: Das
mesh_terms: D000818:Animals; D001120:Arginine / Q000737:chemistry*; D002589:Cestoda / Q000201:enzymology*; D002845:Chromatography; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D002479:Inclusion Bodies / Q000201:enzymology*; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
chemical_list: D011994:Recombinant Proteins; D001120:Arginine; D019764:Phosphoenolpyruvate Carboxykinase (ATP)
keywords: Inclusion bodies; PEPCK; Raillietina echinobothrida; l-Arginine