The Drosophila gamma-glutamyl carboxylase (d gamma C) has substrate recognition properties similar to that of the vertebrate gamma-carboxylase (gamma C), and its carboxylated product yield, in vitro, was shown to be more than that obtained with the human enzyme. However, whether the Drosophila enzyme is able to gamma-carboxylate the human vitamin K-dependent (VKD) proteins, such as the human coagulation factor IX (hFIX), as synthesized in cultured Drosophila cells was not known. To examine this possibility, the Drosophila Schnider (S2) cell line was transfected with a metallothionein promoter-regulated hFIX-expressing plasmid. After induction with copper ion, expression efficiency of the active hFIX was analyzed by performing enzyme-linked immunosorbent assey (ELISA) and coagulation test on the culture supernatant of the transfected S2 cells during 72 h of postinduction. In comparison with Chinese hamster ovary cell line, S2 cells showed higher (similar to 12-fold) expression level of the hFIX. The gamma-carboxylation of the Drosophila-derived hFIX was confirmed by evaluation of the expressed protein, after being precipitated with barium citrate. The biological activity of the S2 cell-derived hFIX indicated the capability of S2 cells to fulfill the required gamma-carboxylation of the expressed hFIX. Coexpression of the human gamma glutamyl carboxylases (h gamma C) was also shown to improve both expression and gamma-carboxylation of the hFIX. This is the first in vivo data to describe the ability of the d gamma C to recognize the human-based propeptide as substrate, which is an essential step for production of biologically active gamma-carboxylated VKD proteins. (c) 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2012