Improvement of the recombinant human coagulation factor IX expression by co-expression of a novel transcript of Drosophi
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ORIGINAL RESEARCH PAPER
Improvement of the recombinant human coagulation factor IX expression by co-expression of a novel transcript of Drosophila c carboxylase in a human cell line Solmaz Moniri Javadhesari
. Alireza Zomorodipour
Received: 26 October 2019 / Accepted: 1 June 2020 Ó Springer Nature B.V. 2020
Abstract Objective Mammalian cells as the main host for production of human proteins are incapable of complete c-carboxylation of over-expressed Vitamin K Dependent (VKD) proteins. The Drosophila c-glutamyl carboxylase (DcC) has been shown to be more efficient than its human counterpart in c-carboxylation of human substrates, in vitro. Considering the Drosophila c-carboxylase (DcC) efficiency, in comparison with its human counterpart, for recognition and c-carboxylation of a human substrate in vitro, we were determined to study the effect of the DcC on the hFIX expression in a mammalian cell line. With this aim, we examined co-expression of the DcC with the hFIX, in a human cell line. Results While the co-expression of a complete DcC cDNA reduced the hFIX expression, a truncated form
of DcC could improve both the expression level (up to 1211 ng/106 cells/ml on the 4th day of post-transfection) and carboxylation of the expressed hFIX, significantly (p \ 0.009). Conclusions Our findings provided evidences for potential of a partial fragment of the DcC for improvement of the c-carboxylation of a human substrate in a mammalian cell. Our experimental data, in accordance with in silico analysis suggested that the DcC C-terminal fragment, with the advantage of a Kozak-like element has the potential of being expressed as a separate internal translation unit, to generate a peptide with appropriate c-carboxylase activity. Keywords Vitamin-K dependent (VKD) proteins Human coagulation factor IX Gamma-carboxylase Drosophila melanogaster
Solmaz Moniri Javadhesari and Alireza Zomorodipour have contributed equally to this work. S. Moniri Javadhesari (&) A. Zomorodipour (&) Department of Molecular Medicine, Institute of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran e-mail: [email protected] A. Zomorodipour e-mail: [email protected] S. Moniri Javadhesari Department of Biology, Faculty of Basic Sciences, Azarbaijan Shahid Madani University, Tabriz, Iran
Introduction Gamma-carboxylation, as an important post-translational modification of Vitamin K-Dependent (VKD) proteins, occurs in the so-called Gla domain of the protein and plays key role in various physiological processes, mostly involved in hemostasis (Bernard et al. 2001; Hedner 2001; Roth et al. 2001). For a VKD protein, such as the human Factor IX (hFIX), an
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18-amino acid (aa) propeptide sequence, N-terminal to the Gla domain, play critical function as recognition site for c-carboxylase (cC) (Furie et al. 1999; Hallgren et al. 2002). A major feature of a VKD protein is its membrane binding activity, which is generated as a re
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