Samira Bahrami1,2,3, Mahmoud Ghaffari2, Alireza Zomorodipour4. 1. Department of Molecular Medicine, Institute of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology, P.O. Box: 14965/161, Tehran, Iran. 2. Department of Biochemistry, Institute of Biochemistry and Biophysics, Tehran University, Tehran, Iran. 3. Biotechnology Department, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 4. Department of Molecular Medicine, Institute of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology, P.O. Box: 14965/161, Tehran, Iran. zomorodi@nigeb.ac.ir.
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.
OBJECTIVE: To compare the effect of pre-propeptide (pre-pro) of the humanprothrombin (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 hFIXpropeptide, 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.
Entities:
Keywords:
Drosophila S2 cell line; Human factor IX (hFIX); Human prothrombin (hPT); Propeptide; γ-Glutamyl carboxylase (GGCX)