Trans-splicing of Cys mutated coagulation factor VIII

To investigate the improving effect of inter-chain disulfide formation on protein trans-splicing, we introduce a Cys point mutation at Tyr⁶⁶⁴ in heavy chain and at Thr¹⁸²⁶ in light chain of B-domain-deleted FVIII (BDD-FVIII).  By co-transfection of COS-7 cell with the two Cys mutated chain genes, the intracellular protein splicing, inter-chain disulfide formation, secreted BDD-FVIII and bioactivity in culture supernatant were observed.  The data showed that a strengthened spliced BDD-FVIII with an inter-chain disulfide detected by Western blotting and an elevated secretion of spliced BDD-FVIII (128 ± 24 ng·mL⁻¹) compared to control (89 ± 15 ng·mL⁻¹), assayed by a sandwich ELISA.  A Coatest was performed to assay the secretion of bioactivity in culture supernatant and shown a much higher value (0.94 ± 0.08 u·mL⁻¹) compared to that of control (0.62 ± 0.15 u·mL⁻¹).  It suggests that inter-chain disulfide formation could improve protein trans-splicing based dual-vector delivery of BDD-FVIII gene providing experimental evidence for ongoing in vivo study.