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Volume 13, Issue 3 (Autumn 2016) |
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The efficiency of secretion and γ-carboxylation of recombinant human factor IX in stable drosophila cells
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S. Khalilzadeh   , J. Vatandoost |
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| Keywords: Key words: Coagulation factor IX, Hemophilia B, gamma-glutamyl carboxylase, GALA peptide |
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Full-Text [PDF 619 kb]
(1159 Downloads)
| Abstract (HTML) (5353 Views)
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Type of Study: Research |
Subject:
Genetis
Published: 2016/09/7
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Full-Text: (2696 Views) |
References :
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Sci J Iran Blood Transfus Organ 2016; 13(3): 233-242
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The efficiency of secretion and γ-carboxylation
of recombinant human factor IX in stable
drosophila cells
Khalilzadeh S.1,Vatandoost J.1
1Hakim Sabzevari University, Sabzevar, Iran
Abstract
Background and Objectives
Human factor IX (hFIX) during maturation in the liver requires carboxylation of glutamic amino acids in the Gla domain, which is involved in its secretion and activity. Due to the deficiency of mammalian expression systems in the secretion and fully γ_carboxylation of recombinant coagulation factors and higher activity of γ_carboxylase enzyme in Drosophila S2system, the present study was performed to evaluate ability of this system in γ_carboxylation, secretion and activity of recombinant hFIX.
Materials and Methods
In this study, following transfection of S2 cells with pMT-hFIX vector, the ELISA and aPTT tests were used to evaluate the expression and activity of recombinant hFIX. In addition, γ_carboxylation of factor IX was approved by barium citrate precipitation. The samples were analyzed on three consecutive days and being repeated three times.
Results
The coagulation results showed the secretion of active recombinant hFIX by stable S2 cells. Quantitative assessment of recombinant hFIX in medium and cell lysis with ELISA showed 94% secretion efficiency. The results of ELISA on precipitated FIX with barium citrate also indicated that about 45% of secreted recombinant hFIX from S2 cells are fully carboxylated.
Conclusions
Activity and barium citrate precipitation of recombinant hFIX confirmed the ability of S2 cells, unlike other insect cells, in the γ_carboxylation of factor IX. So, our results provide convincing evidence that Drosophila γ_carboxylase perform necessary γ_carboxylation required for secretion and coagulation activity of recombinant hFIX.
Key words: Coagulation factor IX, Hemophilia B, gamma-glutamyl carboxylase, GALA peptide
Received: 25 Nov 2015
Accepted: 5 Apr 2016
Correspondence: Vatandoost J., PhD of Molecular Genetics. Assistant Professor of Hakim Sabzevari University.
P.O.Box: 397, Sabzevar, Iran. Tel: (+9851) 44013329; Fax: (+9851) 44013329
E-mail: j.vatan@hsu.ac.ir
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Khalilzadeh S, Vatandoost J. The efficiency of secretion and γ-carboxylation of recombinant human factor IX in stable drosophila cells. Sci J Iran Blood Transfus Organ 2016; 13 (3) :233-242 URL: http://bloodjournal.ir/article-1-1004-en.html
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