Based on observations indicating that the gamma-carboxylase enzyme has a lower affinity for the protein C (PC) propeptide and that the gamma-carboxylase region in the PC propeptide has a higher net... Show moreBased on observations indicating that the gamma-carboxylase enzyme has a lower affinity for the protein C (PC) propeptide and that the gamma-carboxylase region in the PC propeptide has a higher net charge, expression of recombinant chimeric factor IX (FIX) equipped with the PC propeptide was studied. The prepropeptide of FIX was replaced with that of PC by SOEing PCR and after cloning, recombinant pMT-prepro PC/FIX was transfected into insect Drosophila S2 cells. The expression and activity of expressed FIX were analyzed employing antigen and activity analyses 72 h of post-induction with copper. Higher secretion (1.2 fold) and activity (1.6 fold) levels were observed for chimeric prepro- PC/FIX in relation to wild-type FIX. Furthermore, after barium citrate precipitation, the evaluation of fully gamma-carboxylated FIX indicated that more than 51% of the total FIX produced with the PC prepropeptide was fully gamma-carboxylated, representing a substantial improvement (twofold) over a system employing the native FIX propeptide in which 25% of the protein is fully gamma-carboxylated. The data illustrated that the expression of FIX using the PC propeptide led to much higher fully gamma-carboxylated material, which is preferred to FIX constructs tolerating the sequence for the native FIX propeptide expressed in heterologous S2 systems. Show less
