Delineation of Recombinant Cestode Phosphoenolpyruvate Carboxykinase Activity Co-expressed with Molecular Chaperones

Abstract

Phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) of cestodes is considered a possible anthelmintic target because of its differential role in their hosts. In an earlier study, the recombinant PEPCK from Raillietina echinobothrida (rePEPCK) was overexpressed as inclusion bodies and was solubilized following renaturation with chemical additives, specifically L-arginine. Molecular chaperones are alternatives to chemical additives and detergents because they preserve the stability and conformation of the proteins. Hence, in this study, the recombinant rePEPCK was subcloned into the pE-SUMO vector and co-expressed along with the molecular chaperones (e.g. pG-KJE8, pG-Tf2) in Escherichia coli BL21 (DE3) cells. The protein was purified using affinity chromatography and subsequently characterized. The overexpressed rePEPCK was found to be a monomer of ~ 75 kDa. The optimum activity of the enzyme was observed in 50 mM Tris–HCl buffer at pH 7.0. In comparison, Mn²⁺ at 4.0 mM and GDP at 0.6 mM were observed to be the ideal cofactor and nucleotide, respectively. The V_max of the purified rePEPCK was found to be ~ 0.279 U/mg protein and K_m value of ~ 35.87 μM for its substrate. The turnover number (k_cat) of rePEPCK was found to be 4.7 s⁻¹ with catalytic efficiency (k_cat/K_m) 1.31 × 10⁵ M⁻¹ s⁻¹. The chaperones interacted with the key amino acids of PEPCK. This investigation explored the role of the chaperones in producing biologically active rePEPCK for its characterisation and may improve the understanding of the biochemical and biophysical properties of the enzyme as an anthelmintic target.