Expression and identification of recombinant humanuPA_(17)-KPI in Pichia pastoris

Objective To explore the method of secretory expression of recombinant human urokinase-type plasminogen activator-Kunitz protease inhibitor(uPA17-KPI) in Pichia pastoris.Methods On the basis of KPI-pPICZαC,we constructed the rhuPA17-KPI-pPICZαC eukaryon expression vector through gene recombinant technology.The recombinant plasmid was transformed into Pichia pastoris X-33 via electroporation.The transformed positive strains were screened by PCR.The supernatant were analyzed by SDS-PAGE and trypsin inhibition experiment to screen fermentation supernatant.Results After corresponding enzyme cutting identification and DNA sequencing proving,we confirmed that uPA17-KPI-pPICZαC was successfully constructed.SDS-PAGE showed that the relative molecular mass of fermentation product was about 8 800.After induction with methanol for 120 h,the yield of uPA17-KPI could be 50 mg/L.uPA17-KPI had the activity of trypsin inhibition.Conclusion Pichia pastoris can express fusion protein of uPA17-KPI efficiently.