Expression of biologically active human clotting factor IX in Drosophila S2 cells: γ‐carboxylation of a human vitamin K‐dependent protein by the insect enzyme

Abstract

The Drosophila γ-glutamyl carboxylase (dγC) has substrate recognition properties similar to that of the vertebrate γ-carboxylase (γC), 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 γ-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 (≈ 12-fold) expression level of the hFIX. The γ-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 γ-carboxylation of the expressed hFIX. Coexpression of the human γ-glutamyl carboxylases (hγC) was also shown to improve both expression and γ-carboxylation of the hFIX. This is the first in vivo data to describe the ability of the dγC to recognize the human-based propeptide as substrate, which is an essential step for production of biologically active γ-carboxylated VKD proteins.

Bioengineering of differentiated hepatocytes withhuman factor IX-expressing plasmidsin vitro

“…30 Based on our data, the secretion efficiency of the hFIX obtained from expression of different constructs in HepG2 and CHO cells varied between 45-96% and 50-91% respectively (Table 3).…”

Successful synthesis of active human coagulation factor VII by co-expression of mammalian gamma-glutamyl carboxylase and modification of vit.K cycle in Drosophila Schneider S2 cells

“…In a recent study, biologically active human FIX was expressed in S2 cells by transient transfection of the pMT-hFIX expression vector without addition of vit.K. Moreover, co-expression of human GGCX further enhanced human FIX expression (Vatandoost et al 2012). However, some caveats of their study were the presence of FBS in the culture supernatant, transient expression, and absence of vit.K in the medium.…”

“…6). On the other hand, these were unnecessary for the synthesis of biologically active recombinant human FIX (Vatandoost et al 2012). Secretion of recombinant proteins is an important aspect of expression systems.…”

Stable and Transient Expression of Human Coagulation Factor IX in Mammalian Expression Systems; CHO Versus HEK Cells

“…CHO and HEK cells (a kindly gift of Dr. Zomorodipour, NIGEB, Iran) were grown in a 5% CO 2 and 37°C, subcultured at a density of 2 × 10 5 cells in a volume of 2 mL in 6-well plates (13), and transfected with 2 µg pcDNA3-hFIX using the calcium phosphate method. Individual clones were expanded in the presence of 450 µg/mL Geneticin for 2 months.…”

“…Since the current treatment for hemophilia B patients consists of replacement therapy with recombinant factor IX (FIX) (13), selecting a host system for the expression of recombinant proteins should be carefully evaluated. On the other hand, the production of cost-effective and highquality recombinant human FIX would have a remarkable impact on the treatment of hemophilia B patients worldwide.…”