Expression and biological activity of recombinant human coagulation factor X in HEK293 cells

Recombinant human coagulation factor X (rhFX) plays a crucial role in hemostasis and holds potential therapeutic applications for bleeding disorders. This study aimed to establish an efficient expression and purification system for rhFX using HEK293 cells, enhancing its therapeutic potential. Employing molecular cloning and transient transfection, the FX gene was successfully integrated into the pcDNA3.1 vector and transfect to HEK293 cells, yielding high expression levels of rhFX, as confirmed by Western blot analysis and prothrombin time assays. The optimal conditions for protein expression were identified, with peak rhFX concentrations reaching ∼0.005 mg/L and demonstrating procoagulant activity of 5.79 %-6.30 %. Stable HEK293 cell lines were generated, showing consistent expression of rhFX and significantly increased procoagulant activity (61.1 %-78.3 %). Further adaptation to suspension culture improved rhFX yield, achieving concentrations of 13.16 mg/L and enhanced procoagulant activity (658.4 %). Safety assessments confirmed the absence of microbial contamination and genetic fidelity of cell lines. Additionally, the study established a robust screening model for FXa inhibitors, achieving an IC50 value for Edoxaban of 3.56 nM, which aligns with existing literature. Our findings highlight the successful production and biological validation of rhFX, emphasizing its potential for therapeutic applications in coagulation disorders.