Nongkhlaw Joplin, Syngkli Superior, Moirangthem Miranda, Das Bidyadhar
Department of Zoology, NEHU, Shillong, 793022, India.
Protein J. 2025 Jul 11. doi: 10.1007/s10930-025-10279-4.
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, Mn at 4.0 mM and GDP at 0.6 mM were observed to be the ideal cofactor and nucleotide, respectively. The V of the purified rePEPCK was found to be ~ 0.279 U/mg protein and K value of ~ 35.87 μM for its substrate. The turnover number (k) of rePEPCK was found to be 4.7 s with catalytic efficiency (k/K) 1.31 × 10 M s. 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.
由于磷酸烯醇式丙酮酸羧激酶（PEPCK；EC 4.1.1.32）在绦虫及其宿主中发挥着不同作用，因此被认为是一个潜在的驱虫靶点。在早期研究中，来自棘盘瑞利绦虫（Raillietina echinobothrida）的重组PEPCK（rePEPCK）以包涵体形式过量表达，并在使用化学添加剂（特别是L-精氨酸）复性后溶解。分子伴侣可替代化学添加剂和去污剂，因为它们能保持蛋白质的稳定性和构象。因此，在本研究中，重组rePEPCK被亚克隆到pE-SUMO载体中，并与分子伴侣（如pG-KJE8、pG-Tf2）在大肠杆菌BL21（DE3）细胞中共同表达。该蛋白通过亲和层析进行纯化，随后进行表征。发现过量表达的rePEPCK是一种约75 kDa的单体。在pH 7.0的50 mM Tris-HCl缓冲液中观察到该酶的最佳活性。相比之下，4.0 mM的Mn和0.6 mM的GDP分别被认为是理想的辅因子和核苷酸。纯化后的rePEPCK的V约为0.279 U/mg蛋白，其底物的K值约为35.87 μM。rePEPCK的周转数（k）为4.7 s，催化效率（k/K）为1.31×10 M s。分子伴侣与PEPCK的关键氨基酸相互作用。本研究探讨了分子伴侣在产生具有生物活性的rePEPCK以进行表征方面的作用，并可能增进对该酶作为驱虫靶点的生化和生物物理特性的理解。
