Production of recombinant human factor IX by propeptide modification in Drosophila S2 cell line

  • Samira Bahrami
  • Mahmoud Ghaffari
  • Alireza ZomorodipourEmail author
Original Research Paper



To 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.


The 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 γ-carboxylation, reduced the FIX expression efficiency.


Potential of the hPT pre-pro sequence for FIX expression in Drosophila cells, was confronted by γ-glutamyl carboxylase (GGCX) saturation in ER, besides the functional importance of -9 amino acid in propeptide is described; these are noteworthy for production of γ-carboxylated proteins.


Drosophila S2 cell line γ-Glutamyl carboxylase (GGCX) Human factor IX (hFIX) Human prothrombin (hPT) Propeptide 



This study was supported by a grant (Project No. 372) from the National Institute of Genetic Engineering and Biotechnology of Iran.

Supporting information

Supplementary Table 1—List of the used oligonucleotides. Restriction sites are shown in gray. The Drosophila Kozak sequence is underlined.

Author’s contributions

SB, AZ, and MGh: substantial contributions to the conception and design of the work. S.B, and AZ: contributions to the acquisition, analysis and interpretation of data for the study. SB: contributions to drafting the work. SB, AZ: revised the manuscript for important intellectual content. SB: conducted final approval of the version to the published. AZ: was accountable for all aspects of the work. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare no financial or commercial conflict of interest.

Supplementary material

10529_2019_2643_MOESM1_ESM.docx (12 kb)
Supplementary material 1 (DOCX 12 kb)


  1. Bandyopadhyay PK, Garrett JE, Shetty RP, Keate T, Walker CS, Olivera BM (2002) Gamma-glutamyl carboxylation: an extracellular posttranslational modification that antedates the divergence of molluscs, arthropods, and chordates. Proc Natl Acad Sci USA 99:1264–1269. CrossRefGoogle Scholar
  2. Bandyopadhyay PK, Clark K, Stevenson BJ, Rivier JE, Olivera BM, Golic KG, Rong YS (2006) Biochemical characterization of Drosophila gamma-glutamyl carboxylase and its role in fly development. Insect Mol Biol 15:147–156. CrossRefGoogle Scholar
  3. Berkner KL (2000) The vitamin K–dependent carboxylase. J Nutr 130:1877–1880CrossRefGoogle Scholar
  4. Blostein M, Cuerquis J, Landry S, Galipeau J (2008) The carboxylation efficiency of the vitamin K-dependent clotting factors: studies with factor IX. Haemophilia 14:1063–1068CrossRefGoogle Scholar
  5. Bunch TA, Grinblat Y, Goldstein LS (1988) Characterization and use of the Drosophila metallothionein promoter in cultured Drosophila melanogaster cells. Nucleic Acids Res 16:1043–1061CrossRefGoogle Scholar
  6. Camire RM, Larson PJ, Stafford DW, High KA (2000) Enhanced gamma-carboxylation of recombinant factor X using a chimeric construct containing the prothrombin propeptide. Biochemistry 39:14322–14329CrossRefGoogle Scholar
  7. Cavener DR (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res 15:1353–1361CrossRefGoogle Scholar
  8. Galeffi P, Brownlee GG (1987) The propeptide region of clotting factor IX is a signal for a vitamin K dependent carboxylase: evidence from protein engineering of amino acid-4. Nucleic Acids Res 15:9505–9513CrossRefGoogle Scholar
  9. Haddad-Mashadrizeh A, Zomorodipour A, Izadpanah M, Sam MR, Ataei F, Sabouni F, Hosseini SJ (2009) A systematic study of the function of the human β-globin introns on the expression of the human coagulation factor IX in cultured Chinese hamster ovary cells The. J Gene Med 11:941–950CrossRefGoogle Scholar
  10. Johansen H, van der Straten A, Sweet R, Otto E, Maroni G, Rosenberg M (1989) Regulated expression at high copy number allows production of a growth-inhibitory oncogene product in Drosophila Schneider cells. Genes Dev 3:882–889CrossRefGoogle Scholar
  11. Jorgensen MJ, Cantor AB, Furie BC, Brown CL, Shoemaker CB, Furie B (1987) Recognition site directing vitamin K-dependent γ-carboxylation resides on the propeptide of factor IX. Cell 48:185–191CrossRefGoogle Scholar
  12. Kessler CM (2005) New perspectives in hemophilia treatment American Society of. Hematology 2005:429–435CrossRefGoogle Scholar
  13. Khorshidi S, Zomorodipour A, Behmanesh M, Vatandoost J, Bos MH (2015) Functional expression of the human coagulation factor IX using heterologous signal peptide and propeptide sequences in mammalian cell line. Biotechnol Lett 37:1773–1781CrossRefGoogle Scholar
  14. Kim YK, Shin HS, Tomiya N, Lee YC, Betenbaugh MJ, Cha HJ (2005) Production and N-glycan analysis of secreted human erythropoietin glycoprotein in stably transfected Drosophila S2 cells. Biotechnol Bioeng 92:452–461. CrossRefGoogle Scholar
  15. Larson PJ, Stanfield-Oakley SA, VanDusen WJ, Kasper CK, Smith KJ, Monroe DM, High KA (1996) Structural integrity of the-carboxyglutamic acid domain of human blood coagulation Factor IXa is required for its binding to cofactor VIIIa. J Biol Chem 271:3869–3876CrossRefGoogle Scholar
  16. Li T, Yang CT, Jin D, Stafford DW (2000) Identification of a Drosophila vitamin K-dependent gamma-glutamyl carboxylase. J Biol Chem 275:18291–18296. CrossRefGoogle Scholar
  17. Lin PJ, Jin DY, Tie JK, Presnell SR, Straight DL, Stafford DW (2002) The putative vitamin K-dependent gamma-glutamyl carboxylase internal propeptide appears to be the propeptide binding site. J Biol Chem 277:28584–28591. CrossRefGoogle Scholar
  18. Liu J, Jonebring A, Hagström J, Nyström A-C, Lövgren A (2014) Improved expression of recombinant human factor IX by co-expression of GGCX, VKOR and Furin. Protein J 33:174–183CrossRefGoogle Scholar
  19. Mannucci PM, Tuddenham EG (2001) The hemophilias—from royal genes to gene therapy. N Engl J Med 344:1773–1779CrossRefGoogle Scholar
  20. Orlova N, Kovnir S, Vorobiev I, Gabibov A (2012) Coagulation factor IX for hemophilia B therapy. Acta Nat 4:13Google Scholar
  21. Pan LC, Price PA (1985) The propeptide of rat bone gamma-carboxyglutamic acid protein shares homology with other vitamin K-dependent protein precursors. Proc Natl Acad Sci USA 82:6109–6113CrossRefGoogle Scholar
  22. Pipe SW (2008) Recombinant clotting factors. Thromb Haemost 99:840CrossRefGoogle Scholar
  23. Pudota BN, Miyagi M, Hallgren KW, West KA, Crabb JW, Misono KS, Berkner KL (2000) Identification of the vitamin K-dependent carboxylase active site: Cys-99 and Cys-450 are required for both epoxidation and carboxylation. Proc Natl Acad Sci USA 97:13033–13038CrossRefGoogle Scholar
  24. Reveillaud I, Niedzwiecki A, Bensch KG, Fleming JE (1991) Expression of bovine superoxide dismutase in Drosophila melanogaster augments resistance of oxidative stress. Mol Cell Biol 11:632–640CrossRefGoogle Scholar
  25. Schaub RG (2011) Recent advances in the development of coagulation factors and procoagulants for the treatment of hemophilia. Biochem Pharmacol 82:91–98CrossRefGoogle Scholar
  26. Schneider I (1972) Cell lines derived from late embryonic stages of Drosophila melanogaster. J Embryol Exp Morphol 27:353–365Google Scholar
  27. Soejima Y et al (2013) Comparison of signal peptides for efficient protein secretion in the baculovirus-silkworm system. Open Life Sci 8:1–7CrossRefGoogle Scholar
  28. Stanley TB, Wu S-M, Houben RJ, Mutucumarana VP, Stafford DW (1998) Role of the propeptide and γ-glutamic acid domain of factor IX for in vitro carboxylation by the vitamin K-dependent carboxylase. Biochemistry 37:13262–13268CrossRefGoogle Scholar
  29. Stanley TB, Humphries J, High KA, Stafford DW (1999a) Amino acids responsible for reduced affinities of vitamin K-dependent propeptides for the carboxylase. Biochemistry 38:15681–15687CrossRefGoogle Scholar
  30. Stanley TB, Jin DY, Lin PJ, Stafford DW (1999b) The propeptides of the vitamin K-dependent proteins possess different affinities for the vitamin K-dependent carboxylase. J Biol Chem 274:16940–16944CrossRefGoogle Scholar
  31. Tie J-K, Zheng M-Y, Pope RM, Straight DL, Stafford DW (2006) Identification of the N-linked glycosylation sites of vitamin K-dependent carboxylase and effect of glycosylation on carboxylase function. Biochemistry 45:14755–14763CrossRefGoogle Scholar
  32. Vatandoost J, Zomorodipour A, Sadeghizadeh M, Aliyari R, Bos MH, Ataei F (2012) Expression of biologically active human clotting factor IX in Drosophila S2 cells: gamma-carboxylation of a human vitamin K-dependent protein by the insect enzyme. Biotechnol Prog 28:45–51. CrossRefGoogle Scholar
  33. Wajih N, Hutson SM, Owen J, Wallin R (2005) Increased production of functional recombinant human clotting factor IX by baby hamster kidney cells engineered to overexpress VKORC1, the vitamin K 2,3-epoxide-reducing enzyme of the vitamin K cycle. J Biol Chem 280:31603–31607CrossRefGoogle Scholar
  34. Yao S-N, Wilson JM, Nabel EG, Kurachi S, Hachiya HL, Kurachi K (1991) Expression of human factor IX in rat capillary endothelial cells: toward somatic gene therapy for hemophilia B. Proc Natl Acad Sci USA 88:8101–8105CrossRefGoogle Scholar
  35. Zhang L, Leng Q, Mixson AJ (2005) Alteration in the IL-2 signal peptide affects secretion of proteins in vitro and in vivo. J Gene Med 7:354–365CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Samira Bahrami
    • 1
    • 2
    • 3
  • Mahmoud Ghaffari
    • 2
  • Alireza Zomorodipour
    • 1
    Email author
  1. 1.Department of Molecular Medicine, Institute of Medical BiotechnologyNational Institute of Genetic Engineering and BiotechnologyTehranIran
  2. 2.Department of Biochemistry, Institute of Biochemistry and BiophysicsTehran UniversityTehranIran
  3. 3.Biotechnology Department, School of Advanced Technologies in MedicineShahid Beheshti University of Medical SciencesTehranIran

Personalised recommendations