ObjectiveTo compare the effect of pre-propeptide (pre-pro) of the human prothrombin (hPT), with both the native and an R-9N mutant forms of the human factor IX (hFIX) pre-pro on the hFIX carboxylation, in Drosophila cell.ResultsThe three different pre-pro sequences, equipped with Drosophila Kozak, were joined to the mature hFIX cDNA and were subjected to transient expression analysis of hFIX in the S2 Drosophila cells, compared to that of a native hFIX cDNA, with its native Kozak. Replacement of the hFIX pre-pro sequence with that of hPT increased the biological activity of hFIX, significantly. The highest total level of hFIX expression occurred for the native hFIX with the Drosophila Kozak. However, the hFIX secretion efficiency with this construct was less than that of the native hFIX with its native Kozak. The R-9N substitution, in the hFIX propeptide, with no apparent effect on the FIX -carboxylation, reduced the FIX expression efficiency.ConclusionPotential of the hPT pre-pro sequence for FIX expression in Drosophila cells, was confronted by -glutamyl carboxylase (GGCX) saturation in ER, besides the functional importance of -9 amino acid in propeptide is described; these are noteworthy for production of gamma-carboxylated proteins.