Expression and functional characterization of recombinant soluble CD40 Ligand in prokaryotic systems

Objective: To develop an optimized prokaryotic expression system for producing functional soluble CD40 ligand (sCD40L) and characterize its hemostatic activity. Methods: The codon-optimized sCD40L gene (encoding residues 113–261) was synthesized and cloned into pET28a vector via BamHI/XhoI sites. After transformation into E. coli BL21(DE3), soluble expression was induced with 0.5 mM IPTG at 20 °C for 12 h. The recombinant protein was purified using nickel-affinity chromatography and analyzed by SDS-PAGE and Western blot. Biological activity was assessed through in vitro platelet aggregation and in vivo hemostasis assays. Results: The pET28a-sCD40L expression vector was successfully constructed, and the protein purity reached more than 90 %. Western blot confirmed the expected ∼20 kDa sCD40L. Functionally, the recombinant sCD40L enhanced platelet aggregation in a dose-dependent manner, and significantly reduced secondary bleeding time in mice. Conclusion: Our optimized prokaryotic system efficiently produces functional sCD40L without requiring eukaryotic post-translational modifications, providing a valuable tool for studying CD40L-mediated hemostasis and developing potential therapeutic applications.