Construction and Expression of Human Coagulation Factor IX in the Pichia Pastoris

Human Coagulation Factor IX (hFIX) plays a crucial role in the process of human endogenous blood clotting. Patients with deficiency of hFIX will suffer from hemophilia B. In this study, we use Pichia Pastoris to produce recombinant hFIX to cure hemophilia B Human liver hFIX cDNA were amplified by RT-PCR and inserted into pPIC9k vector, to construct pPIC9k-hFIX. The pPIC9k-hFIX were integrated into Pichia Pastoris SMD1168 chromosome by electro-transformation. Then we screened recombinant by PCR analysis and G418 selection to obtain high-copy recombinants. SDS-PAGE and Western Blot analysis show that protein expression volume is up to 100 mg/L. The protein was purifed and concentrated by anion exchange membrane diffusion dialysis. Then we assay activity by using lack coagulation factor IX. The activity calculation by standard curve show that the coagulation factor IX expressed by Pichia Pastoris has similar clotting activity (5.5%) compared to normal human plasma. Thus, expression of hFIX in Pichia Pastoris is of feasibility.