Production of recombinant human factor IX by propeptide modification in Drosophila S2 cell line
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.
KeywordsDrosophila 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.
Supplementary Table 1—List of the used oligonucleotides. Restriction sites are shown in gray. The Drosophila Kozak sequence is underlined.
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.
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