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

To develop an optimized prokaryotic expression system for producing functional soluble CD40 ligand (sCD40L) and characterize its hemostatic activity.

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.

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.

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.