Abstract

Background and Objectives

Human factor IX (hFIX) during maturation in the liver requires carboxylation of glutamic amino acids in the Gla domain, which is involved in its secretion and activity. Due to the deficiency of mammalian expression systems in the secretion and fully γ_carboxylation of recombinant coagulation factors and higher activity of γ_carboxylase enzyme in Drosophila S2system, the present study was performed to evaluate ability of this system in γ_carboxylation, secretion and activity of recombinant hFIX. 

Materials and Methods

In this study, following transfection of S2 cells with pMT-hFIX vector, the ELISA and aPTT tests were used to evaluate the expression and activity of recombinant hFIX. In addition, γ_carboxylation of factor IX was approved by barium citrate precipitation. The samples were analyzed on three consecutive days and being repeated three times.

Results

The coagulation results showed the secretion of active recombinant hFIX by stable S2 cells. Quantitative assessment of recombinant hFIX in medium and cell lysis with ELISA showed 94% secretion efficiency. The results of ELISA on precipitated FIX with barium citrate also indicated that about 45% of secreted recombinant hFIX from S2 cells are fully carboxylated.  

Conclusions

Activity and barium citrate precipitation of recombinant hFIX confirmed the ability of S2 cells, unlike other insect cells, in the γ_carboxylation of factor IX. So, our results provide convincing evidence that Drosophila γ_carboxylase perform necessary γ_carboxylation required for secretion and coagulation activity of recombinant hFIX.