Toward the Development of a Biosimilar Variant of Glucarpidase (Carboxypeptidase G2): Secretory Production, Optimization, and Immobilization.

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^‒1 and 0.0285 UmL^‒1, 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.