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Page 38
Biochemistry & Molecular Biology Journal
ISSN: 2471-8084
Internat i ona l Conference on
Biotechnology, Biomarkers
& Systems Biology
M a r c h 0 4 - 0 5 , 2 0 1 9
Am s t e r d a m , N e t h e r l a n d s
Biotechnology, Biomarkers & Systems Biology 2019
Production of biologically active human vitamin K-dependent proteins
(VKDPs) in heterologous hosts is challenging, due to insufficient
carboxylation. In VKDPs, an N-terminal propeptide containing recognition site
for γ-glutamyl carboxylase (GGCX) is required for carboxylation. The weak-
binding of the propeptide to GGCX, increases the carboxylation rate of the
protein. The human prothrombin (hPT) is highly carboxylated and its propeptide
affinity to GGCX is 10-fold weaker than that of the human factor IX (hFIX). To
study the function of the hPT propeptide on the carboxylation efficiency of
hFIX in Drosophila cell, we generated three constructs, based on a Drosophila-
specific expression vector, carrying a chimeric hFIX cDNA next to the hPT
pre-pro sequence, a mutant hFIX cDNA carrying an R-9N substitution in its
propeptide, and a normal hFIX cDNA. The three constructs were subjected for
transient expression analysis of hFIX in a Drosophila cell line, by performing
coagulation test, ELISA and γ-carboxylation assay, on the cultured media after
various post transfection time. Based on the results obtained, the functional
impact of the hPT propeptide on the hFIX γ-carboxylation, in a distantly related
host, was addressed. Our finding suggested the saturation of Drosophila
GGCX, as a result of accumulation of hFIX in endoplasmic reticulum. These
results demonstrated the functional importance of amino acid at position -9
in a mammalian derived propeptide on the expression efficiency of its cognate
protein.
Biography
Samira Bahrami has completed her MSc in the field of
Biochemistry from Tehran University. Her thesis was about
Studying the expression hFIX, when it’s signal/propetide is
replaced with that of the human propetide. This study was
supported by a grant (Project No. 372) from the National
Institute of Genetic Engineering and Biotechnology (NIGEB)
of Iran. She has worked at the NIGEB as a Research Assistant
for about two years. Now, she is a PhD candidate in Molecular
Medicine at Shahid beheshti University of Medical Sciences,
Tehran, Iran. Her PhD thesis is about Assessment of protein
expression pattern inmuscle-invasive bladder tissue using 2-DE
and MS techniques. This study is performing for the first time in
Iran and is supported by a grant from the National Institute for
Medical Research Development (NIMAD).
Effect of propeptide alteration on the expression of
recombinant Human factor IX in Drosophila S2 cell line
Samira Bahrami1, Alireza Zomorodipour 2 and Maryam Moradi
Chaleshtori1
1Shahid Beheshti University of Medical Sciences, Iran
2IMB-National Institute of Genetic Engineering and Biotechnology, Iran
Samira Bahrami et al., Biochem Mol biol J 2019, Volume:5
DOI: 10.21767/2471-8084-C1-023