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

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

Abstract

Objective

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.

Results

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.

Conclusion

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.

Keywords

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

Notes

Acknowledgements

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)

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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

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