Some recombinant vitamin K-dependent blood coagulation factors (factors VII, IX, and protein C) have become valuable pharmaceuticals in the treatment of bleeding complications and sepsis. Because of their vitamin K-dependent post-translational modification, their synthesis by eukaryotic cells is essential. The eukaryotic cell harbors a vitamin K-dependent γ-carboxylation system that converts the proteins to γ-carboxyglutamic acid-containing proteins. However, the system in eukaryotic cells has limited capacity, and cell lines overexpressing vitamin K-dependent clotting factors produce only a fraction of the recombinant proteins as fully γ-carboxylated, physiologically competent proteins. In this work we have used recombinant human factor IX (r-hFIX)-producing baby hamster kidney (BHK) cells, engineered to stably overexpress various components of the γ-carboxylation system of the cell, to determine whether increased production of functional r-hFIX can be accomplished. All BHK cell lines secreted r-hFIX into serum-free medium. Overexpression of γ-carboxylase is shown to inhibit production of functional r-hFIX. On the other hand, cells overexpressing VKORC1, the reduced vitamin K cofactor-producing enzyme of the vitamin K-dependent γ-carboxylation system, produced 2.9-fold more functional r-hFIX than control BHK cells. The data are consistent with the notion that VKORC1 is the rate-limiting step in the system and is a key regulatory protein in synthesis of active vitamin K-dependent proteins. The data suggest that overexpression of VKORC1 can be utilized for increased cellular production of recombinant vitamin K-dependent proteins. Some recombinant vitamin K-dependent blood coagulation factors (factors VII, IX, and protein C) have become valuable pharmaceuticals in the treatment of bleeding complications and sepsis. Because of their vitamin K-dependent post-translational modification, their synthesis by eukaryotic cells is essential. The eukaryotic cell harbors a vitamin K-dependent γ-carboxylation system that converts the proteins to γ-carboxyglutamic acid-containing proteins. However, the system in eukaryotic cells has limited capacity, and cell lines overexpressing vitamin K-dependent clotting factors produce only a fraction of the recombinant proteins as fully γ-carboxylated, physiologically competent proteins. In this work we have used recombinant human factor IX (r-hFIX)-producing baby hamster kidney (BHK) cells, engineered to stably overexpress various components of the γ-carboxylation system of the cell, to determine whether increased production of functional r-hFIX can be accomplished. All BHK cell lines secreted r-hFIX into serum-free medium. Overexpression of γ-carboxylase is shown to inhibit production of functional r-hFIX. On the other hand, cells overexpressing VKORC1, the reduced vitamin K cofactor-producing enzyme of the vitamin K-dependent γ-carboxylation system, produced 2.9-fold more functional r-hFIX than control BHK cells. The data are consistent with the notion that VKORC1 is the rate-limiting step in the system and is a key regulatory protein in synthesis of active vitamin K-dependent proteins. The data suggest that overexpression of VKORC1 can be utilized for increased cellular production of recombinant vitamin K-dependent proteins. Recombinant factors VII, VIIa, VIII, IX, and protein C have become important pharmaceuticals in treatment of traumatic bleeding complications (1Hedner U. Semin. Hematol. 2001; 38: 43-47Crossref PubMed Scopus (69) Google Scholar), hemophilia (2Roth D.A. Kessler C.M. Pasi J. Rup B. Courter S.G. Tubridy K.L. Blood. 2001; 98: 3600-3606Crossref PubMed Scopus (162) Google Scholar), and sepsis (3Berbard G.R. Vincent J-L. Laterre P-F. LaRosa S.P. Dhainaut J-F. Lopez-Rodriguez A. Steingrub J.S. Garber G.E. Helterbrand J.D. Ely E.W. Fisher C.J. New Engl. J. Med. 2001; 344: 699-709Crossref PubMed Scopus (5145) Google Scholar). These proteins are parts of the coagulation system (4Dahlback B. J. Intern. Med. 2005; 257: 209-223Crossref PubMed Scopus (138) Google Scholar), a system of plasma and membrane proteins that are responsible for arrest of blood loss at sites of injury. Factors VII, IX, and protein C belong to a family of vitamin K-dependent proteins that are modified post-translationally to contain γ-carboxyglutamic acid (Gla), 1The abbreviations used are: Gla, γ-carboxyglutamic acid; r-hFIX, recombinant human factor IX; VKOR, vitamin K 2,3-epoxide reductase; VKORC1, a putative subunit of VKOR (12Rost S. Fregin A. Ivaskevicius V. Conzelmann E. Hortnagel K. Pelz H.J. Lappegard K. Seifried E. Scharrer I. Tuddenham E.G. Muller C.R. Strom T.M. Oldenburg J. Nature. 2004; 427: 537-541Crossref PubMed Scopus (1006) Google Scholar); ER, endoplasmic reticulum; BHK, baby hamster kidney; TBS, Tris-buffered saline; HPLC, high performance liquid chromatography. Ca2+ binding amino acid residues (5Wallin R. Hutson S.M. Trends Mol. Med. 2004; 10: 299-302Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The modification is carried out by the vitamin K-dependent γ-carboxylation system located in the endoplasmic reticulum (ER) (5Wallin R. Hutson S.M. Trends Mol. Med. 2004; 10: 299-302Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Two essential enzymes of the system are 1) the vitamin K-dependent γ-carboxylase, an integral membrane protein of 92 kDa that requires reduced vitamin K (vit.K1H2) as cofactor, and 2) the warfarin-sensitive enzyme vitamin K 2,3-epoxide reductase (VKOR), which produces the cofactor (5Wallin R. Hutson S.M. Trends Mol. Med. 2004; 10: 299-302Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). A major problem with production of recombinant vitamin K-dependent coagulation factors for use as pharmaceuticals has been poor recovery of functional proteins from the cell medium (6Berkner K.L. Methods Enzymol. 1993; 222: 450-480Crossref PubMed Scopus (64) Google Scholar, 7Kaufman R.J. Wasley L.C. Furie B.C. Furie B. Shoemaker C.B. J. Biol. Chem. 1986; 261: 9622-9628Abstract Full Text PDF PubMed Google Scholar). It has been shown that poor recovery results from incomplete γ-carboxylation of the secreted proteins (7Kaufman R.J. Wasley L.C. Furie B.C. Furie B. Shoemaker C.B. J. Biol. Chem. 1986; 261: 9622-9628Abstract Full Text PDF PubMed Google Scholar) and incomplete removal of the propeptide by the protease PACE/furin in the Golgi apparatus (8Wasley L.C. Rehemtulla A. Bristol J.A. Kaufman R.J. J. Biol. Chem. 1993; 268: 8458-8465Abstract Full Text PDF PubMed Google Scholar). Incomplete γ-carboxylation is a significant problem as <10% of the secreted recombinant vitamin K-dependent proteins have been reported to be fully γ-carboxylated and functional (7Kaufman R.J. Wasley L.C. Furie B.C. Furie B. Shoemaker C.B. J. Biol. Chem. 1986; 261: 9622-9628Abstract Full Text PDF PubMed Google Scholar). It is believed that incomplete γ-carboxylation occurs when an excess of newly synthesized precursors of vitamin K-dependent proteins appears in the ER and exceeds the capacity of the cell's γ-carboxylation system to fully modify all of the precursors (6Berkner K.L. Methods Enzymol. 1993; 222: 450-480Crossref PubMed Scopus (64) Google Scholar, 7Kaufman R.J. Wasley L.C. Furie B.C. Furie B. Shoemaker C.B. J. Biol. Chem. 1986; 261: 9622-9628Abstract Full Text PDF PubMed Google Scholar). Following cloning of γ-carboxylase (9Wu S.M. Cheung W.F. Frazier D. Stafford D.W. Science. 1991; 254: 1634-1636Crossref PubMed Scopus (178) Google Scholar), it was shown, surprisingly, that overexpression of γ-carboxylase in cells stably expressing recombinant human factor IX (r-hFIX) inhibited synthesis of functional r-hFIX (10Rehemtulla A. Roth D.A. Wasley L.C. Kuliopulos A. Walsh C.T. Furie B. Furie B.C. Kaufman R.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4611-4615Crossref PubMed Scopus (65) Google Scholar). Because of our limited knowledge of the vitamin K-dependent γ-carboxylation system, the mechanism of inhibition is not fully understood (11Hallgren K.W. Hommema E.L. McNally B.A. Berkner K.L. Biochemistry. 2002; 41: 15045-15055Crossref PubMed Scopus (46) Google Scholar). However, the recent identification of VKORC1 (12Rost S. Fregin A. Ivaskevicius V. Conzelmann E. Hortnagel K. Pelz H.J. Lappegard K. Seifried E. Scharrer I. Tuddenham E.G. Muller C.R. Strom T.M. Oldenburg J. Nature. 2004; 427: 537-541Crossref PubMed Scopus (1006) Google Scholar), an 18-kDa ER membrane protein believed to be a subunit of VKOR (13Cain D. Hutson S.M. Wallin R. J. Biol. Chem. 1997; 272: 29068-29075Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), has provided new opportunities to understand the system. When expressed in various cell lines, VKORC1 increases reduced vitamin K cofactor production (14Wajih N. Sane D.C. Hutson S.M. Wallin R.J. J. Biol. Chem. 2005; 280: 10540-10547Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 15Li T. Chang C.Y. Jin D.Y. Lin P.J. Khvorova A. Stafford D.W. Nature. 2004; 427: 541-544Crossref PubMed Scopus (608) Google Scholar). We have shown that overexpression of VKORC1 in baby hamster kidney (BHK) cells increases the capacity of the cell's γ-carboxylation system (14Wajih N. Sane D.C. Hutson S.M. Wallin R.J. J. Biol. Chem. 2005; 280: 10540-10547Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Furthermore, and importantly, we have shown that VKOR is the rate-limiting step in the system (14Wajih N. Sane D.C. Hutson S.M. Wallin R.J. J. Biol. Chem. 2005; 280: 10540-10547Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 16Wallin R. Sane D.C. Hutson S.M. Thromb. Res. 2002; 108: 221-226Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). This finding is consistent with results obtained by Hallgren et al. (11Hallgren K.W. Hommema E.L. McNally B.A. Berkner K.L. Biochemistry. 2002; 41: 15045-15055Crossref PubMed Scopus (46) Google Scholar). In a previous article we measured increased γ-carboxylation capacity by VKORC1-transfected cells as γ-carboxylation of the synthetic γ-carboxylase peptide substrate FLEEL. In this report, we have shown that r-hFIX-producing BHK cells, stably transfected with a VKORC1 cDNA construct, produce 2.9-fold more fully γ-carboxylated functional r-hFIX than r-hFIX-producing BHK cells using the endogenous vitamin K-dependent system of the cell to γ-carboxylate the protein. This finding provides new opportunities for increased production of fully γ-carboxylated and functional recombinant vitamin K-dependent proteins. Engineering of BHK 21 cell lines stably overexpressing γ-carboxylase, VKORC1, and VKORC1 + γ-carboxylase is described in detail in Ref. 14Wajih N. Sane D.C. Hutson S.M. Wallin R.J. J. Biol. Chem. 2005; 280: 10540-10547Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar. BHK cells were washed with ice-cold phosphate-buffered saline, and cell protein was extracted with 50 mm Tris, 1% Nonidet P-40, 0.25% sodium-deoxycholate, 150 mm NaCl, 1 mm EDTA, pH 7.2, containing 10 μg/ml of the protease inhibitor mixture for use with mammalian cell and tissue extracts (Sigma) as described (14Wajih N. Sane D.C. Hutson S.M. Wallin R.J. J. Biol. Chem. 2005; 280: 10540-10547Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). SDS-PAGE was carried out on 8–16% Criterion gradient gels (BioRad). Western blotting was carried out as described (14Wajih N. Sane D.C. Hutson S.M. Wallin R.J. J. Biol. Chem. 2005; 280: 10540-10547Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Furin Convertase antibodies (PA1–062) were from Affinity BioReagents, Golden, CO. Plasmid pCMV5FIX containing the full-length human factor IX (hFIX) cDNA was purchased from American Type Culture Collection (catalogue number 79871). BamHI restriction sites were generated at the 5′- and 3′-ends of hFIX cDNA using PCR to clone the cDNA into the pLXIN retroviral vector (Clontech) for expression in mammalian cell lines. Kozak sequence was also generated at the 5′-end of the hFIX cDNA. The oligonucleotides used for hFIX PCR were sense primer 5′-GGA TCC GCC GCC ACC ATG CAG CGC GTG AAC ATG ATC-3′ and antisense primer 5′–GGA TCC CTT TCA TTA AGT GAG CTT TG-3′. Thirty-five cycles of PCR were performed under the following conditions: Denature at 94 °C for 30 s, anneal for 1 min at 55 °C, followed by an extension at 72 °C for 7 min. The modified cDNA for human factor IX was then cloned into the BamHI site of the pLXIN vector under control of the cytomegalovirus promoter. Recombinant plasmid with cDNA for hFIX was sequenced on both strands to eliminate any PCR errors. PG 13 cells (a kind gift from Dr. Charles Morrow, Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, NC) were transfected with the pLXIN retroviral vector containing the hFIX insert for packing. Retroviral particles were harvested from the PG 13 cell medium 72 h post-transfection. Harvested medium was centrifuged at 3000 × g to remove cell debris. The supernatant was collected and used to infect BHK cells and BHK cells engineered to overexpress γ-carboxylase, VKORC1, and VKORC1 + γ-carboxylase. Stable r-hFIX-producing cell lines were selected with G418 pressure. Cells were seeded and grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 500 μg/ml G418, and 400 μg/ml Zeocin. At confluency, the attached cells were washed twice with PBS and continued growing in Dulbecco's modified Eagle's medium without serum but with the addition of 5 μg/ml vitamin K1 (AquaMEPHYTON; Merck & Co. Inc.). h the serum-free medium was collected and used for of r-hFIX. human factor IX was purchased from and used for production as described R. Scopus Google Scholar). The of antibodies in the was by PubMed Scopus Google Scholar). All the of The hFIX was with and proteins were collected by at × g for 10 min in a The protein was washed with Tris-buffered containing 5 mm and in the with The serum proteins were a an with hFIX as The was in The was by hFIX to acid as by the antibodies that the of the in hFIX were from the with 50 mm in Following of the the was washed with antibodies were with pH containing and pH as described by our R. Scopus Google Scholar). of antibodies were at °C in and as at of r-hFIX and plasma hFIX were with the from determine the of r-hFIX, a of of plasma hFIX was of hFIX was as the of hFIX in of human was carried out by the at Wake Forest University School of of r-hFIX was carried of the protein in under for h at were by to a modification of the described by and D.A. J. PubMed Scopus Google Scholar), which provides of the of Gla, and of the number of residues in r-hFIX was on the of and residues in hFIX K. E.W. J. PubMed Scopus Google Scholar). was by from a × (Sigma) with a mixture of of A pH 7.2, containing and and of to amino acid of amino acid and protein was with of a of acid in by of containing g of acid with of in and the to with of C and by 50 of in of to which of pH 50 of and of were were and for min in the system of the of was with a with and of and of protein on were with to determine the the protein of r-hFIX protein of Western with human were and with All were to be in the of the Stable r-hFIX and Recombinant of the K-dependent Retroviral particles containing an hFIX cDNA were used to infect BHK cells and BHK cells stably overexpressing 1) VKORC1, 2) γ-carboxylase, and VKORC1 + γ-carboxylase. Engineering of cell lines and is described in Ref. 14Wajih N. Sane D.C. Hutson S.M. Wallin R.J. J. Biol. Chem. 2005; 280: 10540-10547Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar. BHK cells and the BHK cell lines and produced + and of cells, cell r-hFIX production by cell was to be by overexpression of proteins of the γ-carboxylation system. r-hFIX that was to the of fully γ-carboxylated functional plasma factor IX (7Kaufman R.J. Wasley L.C. Furie B.C. Furie B. Shoemaker C.B. J. Biol. Chem. 1986; 261: 9622-9628Abstract Full Text PDF PubMed Google Scholar), was from the cell medium by on a with hFIX antibodies as in the shown in of r-hFIX were by and from 1 with 50 mm Recombinant hFIX proteins that were to this in the fraction from These of r-hFIX were on an 2) with hFIX antibodies as were from the with pH which in We by Western blotting with hFIX antibodies that the fraction from not contain any This finding that the in 1 all r-hFIX from the cell medium that was the SDS-PAGE of proteins in 1 and with human plasma factor IX is shown in All proteins as a with an of of functional and r-hFIX. in 1 and shown in 1 from cell medium obtained from BHK cells expressing r-hFIX and VKORC1 were to SDS-PAGE in a 8–16% Criterion proteins in 1 and are shown with human plasma that of 1 with fully γ-carboxylated we plasma hFIX on 1 under the as we r-hFIX from the cell medium in the of the plasma was by and from the with 50 mm the was with pH to any proteins on the proteins in a Western of plasma with the hFIX antibodies and Western of proteins in 1 and shown, both the and the antibodies r-hFIX in the 1 but not any hFIX in the pH This that fully γ-carboxylated and functional hFIX was and with from the that and γ-carboxylated r-hFIX not to the we used the pH obtained from medium harvested from BHK cells overexpressing VKORC1 + r-hFIX as a for of r-hFIX of r-hFIX in this pH fraction an of of r-hFIX. When on 1 1) in the of of the proteins in the pH fraction to the that the in 1 provided 1) the we to fully γ-carboxylated r-hFIX and 2) an in to the capacity of our cell lines to produce fully γ-carboxylated of r-hFIX r-hFIX produced by the and production of BHK r-hFIX in a new of r-hFIX by Affinity that on 1 1) in of functional r-hFIX, the protein obtained from cell medium was for clotting and sequence was carried out on the protein from the medium that the of functional r-hFIX. the r-hFIX the of r-hFIX The under the data obtained with r-hFIX by on 1 When with the r-hFIX from all cell lines the as human plasma hFIX the of The the of plasma which is of protein K. E.W. J. PubMed Scopus Google Scholar). cycles of were carried out on r-hFIX from BHK cells overexpressing VKORC1, which the of functional r-hFIX. The sequence was consistent with the sequence of human plasma K. E.W. J. PubMed Scopus Google Scholar). sequence the of the hFIX propeptide in precursors of hFIX be These that the Golgi PACE/furin responsible for removal of the propeptide (8Wasley L.C. Rehemtulla A. Bristol J.A. Kaufman R.J. J. Biol. Chem. 1993; 268: 8458-8465Abstract Full Text PDF PubMed Google Scholar), was active in the BHK cell lines. PACE/furin was by Western blotting in extracts of the BHK cell as a protein consistent with the reported by et al. A. U. V. A. B. Fisher PubMed Google Scholar) for the active protease responsible for factor propeptide The new and important data from this work are in and the in production of functional r-hFIX by cell lines engineered to overexpress proteins of the γ-carboxylation system. with by (10Rehemtulla A. Roth D.A. Wasley L.C. Kuliopulos A. Walsh C.T. Furie B. Furie B.C. Kaufman R.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4611-4615Crossref PubMed Scopus (65) Google Scholar), BHK cells overexpressing γ-carboxylase the production of functional r-hFIX. BHK cell lines engineered to overexpress VKORC1 and VKORC1 + γ-carboxylase produced more active r-hFIX than BHK cells expressing r-hFIX. The VKORC1 overexpressing cell was the of functional r-hFIX and produced 2.9-fold more functional r-hFIX than BHK cells stably expressing r-hFIX These data are consistent with the notion that VKOR is the rate-limiting step in the vitamin K-dependent γ-carboxylation system responsible for post-translational modification of vitamin K-dependent proteins. This is the to production of a recombinant vitamin K-dependent protein by cell lines engineered to a vitamin K-dependent γ-carboxylation system with capacity for post-translational modification (14Wajih N. Sane D.C. Hutson S.M. Wallin R.J. J. Biol. Chem. 2005; 280: 10540-10547Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Because of the work on cellular production of r-hFIX carried out by (6Berkner K.L. Methods Enzymol. 1993; 222: 450-480Crossref PubMed Scopus (64) Google Scholar, 7Kaufman R.J. Wasley L.C. Furie B.C. Furie B. Shoemaker C.B. J. Biol. Chem. 1986; 261: 9622-9628Abstract Full Text PDF PubMed Google Scholar, A. Roth D.A. Wasley L.C. Kuliopulos A. Walsh C.T. Furie B. Furie B.C. Kaufman R.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4611-4615Crossref PubMed Scopus (65) Google Scholar), we on r-hFIX as we our data to In a recent article (14Wajih N. Sane D.C. Hutson S.M. Wallin R.J. J. Biol. Chem. 2005; 280: 10540-10547Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), we of BHK cells stably overexpressing 1) VKORC1, the reduced vitamin K cofactor-producing enzyme of the vitamin K-dependent γ-carboxylation system, 2) γ-carboxylase, the enzyme in the system, and a of VKORC1 + γ-carboxylase with a BHK cells were on (6Berkner K.L. Methods Enzymol. 1993; 222: 450-480Crossref PubMed Scopus (64) Google Scholar) and our data that BHK cells have an endogenous γ-carboxylation system with that is the of this measured in a of other cell lines. In the previous (14Wajih N. Sane D.C. Hutson S.M. Wallin R.J. J. Biol. Chem. 2005; 280: 10540-10547Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), γ-carboxylation was measured in as into the synthetic γ-carboxylase peptide substrate FLEEL. we have whether our BHK cells with engineered γ-carboxylation production of recombinant vitamin K-dependent proteins. a retroviral vector the cDNA for we BHK cells and our engineered BHK cell lines and selected for cell lines stably expressing r-hFIX. The of r-hFIX has been shown to cell lines expressing r-hFIX (6Berkner K.L. Methods Enzymol. 1993; 222: 450-480Crossref PubMed Scopus (64) Google Scholar, 7Kaufman R.J. Wasley L.C. Furie B.C. Furie B. Shoemaker C.B. J. Biol. Chem. 1986; 261: 9622-9628Abstract Full Text PDF PubMed Google Scholar). increased Kaufman et al. (7Kaufman R.J. Wasley L.C. Furie B.C. Furie B. Shoemaker C.B. J. Biol. Chem. 1986; 261: 9622-9628Abstract Full Text PDF PubMed Google Scholar) were to produce cell lines with production than our BHK cell lines. However, their r-hFIX-producing cells not increased production of active r-hFIX (7Kaufman R.J. Wasley L.C. Furie B.C. Furie B. Shoemaker C.B. J. Biol. Chem. 1986; 261: 9622-9628Abstract Full Text PDF PubMed Google Scholar). It was also shown (10Rehemtulla A. Roth D.A. Wasley L.C. Kuliopulos A. Walsh C.T. Furie B. Furie B.C. Kaufman R.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4611-4615Crossref PubMed Scopus (65) Google Scholar) that propeptide was which be for by the cDNA for the protease PACE/furin in the Golgi It that the vitamin K-dependent γ-carboxylation system is a significant rate-limiting step in eukaryotic cell production of functional r-hFIX (7Kaufman R.J. Wasley L.C. Furie B.C. Furie B. Shoemaker C.B. J. Biol. Chem. 1986; 261: 9622-9628Abstract Full Text PDF PubMed Google Scholar). We in this work and in (14Wajih N. Sane D.C. Hutson S.M. Wallin R.J. J. Biol. Chem. 2005; 280: 10540-10547Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 16Wallin R. Sane D.C. Hutson S.M. Thromb. Res. 2002; 108: 221-226Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) that VKOR, the reduced vitamin K cofactor-producing enzyme of the γ-carboxylation system, is the rate-limiting sequence of active r-hFIX in 1 1 and 2) that the functional clotting factor was of precursors that not been by This finding was as it has been shown J.A. Furie B.C. Furie B. Biochemistry. PubMed Scopus Google Scholar) that fully γ-carboxylated precursors of r-hFIX the and the by our antibodies used to functional r-hFIX. The of precursors that PACE/furin in the BHK cells used for our the capacity to r-hFIX secreted at an of cells. However, r-hFIX precursors have been in from 1 in this fraction were not to protein The new and important finding in this work is increased production of functional r-hFIX by BHK cells engineered to overexpress A 2.9-fold in production is has been reported (7Kaufman R.J. Wasley L.C. Furie B.C. Furie B. Shoemaker C.B. J. Biol. Chem. 1986; 261: 9622-9628Abstract Full Text PDF PubMed Google Scholar). the described in this be used to production of r-hFIX for use as a in hemophilia treatment (2Roth D.A. Kessler C.M. Pasi J. Rup B. Courter S.G. Tubridy K.L. Blood. 2001; 98: 3600-3606Crossref PubMed Scopus (162) Google Scholar). engineered cell lines also be used for production of recombinant factor and protein C (1Hedner U. Semin. Hematol. 2001; 38: 43-47Crossref PubMed Scopus (69) Google Scholar, G.R. Vincent J-L. Laterre P-F. LaRosa S.P. Dhainaut J-F. Lopez-Rodriguez A. Steingrub J.S. Garber G.E. Helterbrand J.D. Ely E.W. Fisher C.J. New Engl. J. Med. 2001; 344: 699-709Crossref PubMed Scopus (5145) Google Scholar). in this work and by (10Rehemtulla A. Roth D.A. Wasley L.C. Kuliopulos A. Walsh C.T. Furie B. Furie B.C. Kaufman R.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4611-4615Crossref PubMed Scopus (65) Google Scholar), cells overexpressing recombinant γ-carboxylase produce functional r-hFIX. Hallgren et al. (11Hallgren K.W. Hommema E.L. McNally B.A. Berkner K.L. Biochemistry. 2002; 41: 15045-15055Crossref PubMed Scopus (46) Google Scholar) have that an excess of γ-carboxylase in the ER of γ-carboxylated proteins by with the vitamin K-dependent protein It appears from our data that overexpression of γ-carboxylase is not for increased production of recombinant vitamin K-dependent proteins. The endogenous γ-carboxylase in the ER has a high capacity for γ-carboxylation (14Wajih N. Sane D.C. Hutson S.M. Wallin R.J. J. Biol. Chem. 2005; 280: 10540-10547Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), but this requires of shown, increased cofactor can be provided by overexpressing recombinant However, to fully the capacity of the γ-carboxylation system in engineered cells, a of the system at the be We for with coagulation
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