Moradbeygi Fatemeh, Ghoshoon Mohammad Bagher, Sadeghian Issa, Moradi Anahita, Ghasemi Younes, Morowvat Mohammad Hossein, Hemmati Shiva
Student Research Committee, Shiraz University of Medical Sciences, Shiraz, Iran.
Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
Mol Biotechnol. 2025 Jun 4. doi: 10.1007/s12033-025-01450-4.
Carboxypeptidase G2 (CPG2) detoxifies high-dose methotrexate in cancer or autoimmune therapies. Additionally, CPG2 activates prodrugs at the tumor site in engineered CAR-T cell therapy. Since CPG2 cleaves glutamate from the substrate, it also has potential applications in enhancing food flavors and developing biosensors. However, cytoplasmic expression of recombinant CPG2 often leads to inclusion body formation, necessitating secretory production to improve product quality and streamline downstream industrial processes. This study aimed to perform a comprehensive in silico analysis to identify an optimal signal peptide for CPG2 secretion. The pelB leader sequence was selected for excretory production using the pET22b vector in Escherichia coli. The enzyme was successfully secreted into both the periplasmic space of bacterial cells and the culture medium with activities of about 0.038 UmL and 0.0285 UmL, respectively. Optimal expression conditions were achieved at 25 °C with 0.5-mM IPTG, leading to enhanced yields. In the second step, we aimed to optimize the culture media composition. Therefore, a statistical design of experiments (DOE) approach in Modde software was conducted, evaluating eleven variables via a fractional factorial design (FFD). A central composite face (CCF) design was applied to establish an empirical model for maximizing periplasmic CPG2 production. The final aim was to immobilize the whole E. coli cells in calcium alginate beads for higher metabolic activity, improved growth rates, and plasmid stability. Engineered bacterial cells were entrapped in a mixture of sodium alginate and calcium chloride solution and incubated at 4 °C, 400 rpm for 60 min. Immobilization of CPG2-producing E. coli cells in 2% (w/v) calcium alginate beads yielded spherical beads, maintaining enzyme stability for 10 days, with peak activity on day 5. Conclusively, improved CPG2 solubility, facilitated protein purification, and enzyme stability over multiple reaction cycles offer a cost-effective solution for industrial purposes.
羧肽酶G2（CPG2）可在癌症或自身免疫疗法中使高剂量甲氨蝶呤解毒。此外，在工程化嵌合抗原受体T细胞（CAR-T）疗法中，CPG2可在肿瘤部位激活前药。由于CPG2能从底物上切割下谷氨酸，它在增强食品风味和开发生物传感器方面也具有潜在应用。然而，重组CPG2的胞质表达常常导致包涵体形成，因此需要进行分泌表达以提高产品质量并简化下游工业流程。本研究旨在进行全面的计算机模拟分析，以确定用于CPG2分泌的最佳信号肽。使用pET22b载体在大肠杆菌中选择pelB前导序列进行分泌表达。该酶成功分泌到细菌细胞的周质空间和培养基中，活性分别约为0.038 U/mL和0.0285 U/mL。在25℃和0.5 mM异丙基-β-D-硫代半乳糖苷（IPTG）条件下实现了最佳表达条件，从而提高了产量。在第二步中，我们旨在优化培养基组成。因此，在Modde软件中采用实验设计（DOE）方法，通过分式析因设计（FFD）评估11个变量。应用中心复合表面（CCF）设计建立了一个用于最大化周质CPG2产量的经验模型。最终目标是将整个大肠杆菌细胞固定在海藻酸钙珠中，以实现更高的代谢活性、更快的生长速率和质粒稳定性。将工程化细菌细胞包埋在海藻酸钠和氯化钙溶液的混合物中，于4℃、400 rpm孵育60分钟。将产生CPG2的大肠杆菌细胞固定在2%（w/v）海藻酸钙珠中可得到球形珠，酶稳定性维持10天，第5天活性达到峰值。总之，提高CPG2的溶解度、促进蛋白质纯化以及在多个反应循环中保持酶稳定性，为工业用途提供了一种经济高效的解决方案。
