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.
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.
Department of Genetics, Tarbiat Modares University, Tehran, Iran.
Jafar
J
Vatandoost
Alireza
A
Zomorodipour
Majid
M
Sadeghizadeh
Roghayeh
R
Aliyari
Mettine H A
MH
Bos
Fariba
F
Ataei
D000818:Animals; D001777:Blood Coagulation; D016466:CHO Cells; D019735:Carbon-Carbon Ligases / Q000378:metabolism; D006224:Cricetinae; D004330:Drosophila / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D005164:Factor IX / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014162:Transfection; D014812:Vitamin K / Q000378:metabolism*
D011994:Recombinant Proteins; D014812:Vitamin K; D005164:Factor IX; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase