Effect of prepropeptide replacement on γ-carboxylation and activity of recombinant coagulation factor IX

Abstract

Based on observations indicating that γ-carboxylase enzyme has lesser affinity for propeptide of protein C (PC) and a higher net charge of γ-carboxylase region in PC propeptide, expression of recombinant chimeric Factor IX (FIX) equipped with PC propeptide was studied. The prepropeptide of FIX was replaced with PC by SOEing PCR and after cloning, recombinant pMT-prepro PC/FIX were transfected into the insect Drosophila S2 cells. The expression and activity of the active hFIX were analyzed by performing ELISA and coagulation test during 72 h of post-induction with copper ion. The results showed the higher secretion and activity level with an average of 1.2 and 1.6-fold, respectively, for chimeric prepro- PC/FIX construct in relation to normal FIX. Furthermore, after being precipitated with barium citrate, the evaluation of fully γ-carboxylated FIX indicated that more than 51% of the total FIX produced with the PC prepropeptide was fully γ-carboxylated, represents a substantial improvement (2 fold) over a system employing the native FIX propeptide, by which 25% of the protein is fully γ-carboxylated. The data illustrated that the expression of FIX using the PC propeptide led to much higher fully γ-carboxylated material, which is preferred to FIX constructs tolerating the sequence for the native FIX propeptide expressed in heterologous S2 systems.