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|Εξειδίκευση τύπου : ||Περίληψη σε συνέδριο|
|Τίτλος: ||Directed mutagenesis on the gene of coagulation factor IX and their effect on the proteolytic activity|
|Δημιουργός/Συγγραφέας: ||Iskas, M. S.|
Papi, R. M.
Rafailidis, S. P.
Makris, P. E.
[EL] Κυριακίδης, Δ.Α.[EN] Kyriakidis, D.A.
|Εκδότης: ||Springer Wien|
|Περίληψη: ||In this study site-directed mutagenesis was performed on coagulation factor IX (fIX) gene and their effect on the protein’s proteolytic activity was studied. Blood coagulation factor IX (fIX) is a vitamin K-dependent plasma serine protease that plays a major role in coagulation mechanism and haemostasis. It is synthesized in hepatocytes as a precursor protein of 461 amino acids. After several post-translational modifications the mature protein is secreted in the circulation as inactive zymogen. The zymogen is then activated during the coagulation cascade into an active serine protease. A truncated form of fIX gene (rf9) inserted in a pET22b vector was used in our experiments so as to be able to overexpress the protein in prokaryotic cells. Two mutations were induced on this gene. Both of them are described in databases as polymorphisms. The first one (A21975G) is on the protein’s activation peptide and causes the substitution of Thr194 by an Ala residue. The prevalence of this G containing allele in general population is 23%. However in our previous study we found that in a certain group of thrombophilic patients the prevalence of the G containing allele was 50%. The second mutation (A32881C) is also described as polymorphism and causes the substitution of Thr461 by a Gly residue and involves the 2% of the population. Both mutated and native forms of the truncated coagulation factor IX (rf9) were overexpressed in E. coli BL21 cells after induction with IPTG. The truncated factor rf9 lacks its first N-terminal domains; therefore it doesn’t require posttranslational modifications in order to gain activity. Proteolytic activity of rf9 was studied using an amidolytic assay. Rf9 was first activated using Russell’s viper venom FX activating protein (RVV-X). The specific tripeptide MSD- Phe-Gly-Arg-pNA was used as a substrate. Proteins were purified from the cytoplasm using a Q-Sepharose ff column. They were activated and their proteolytic activity was studied. A difference in the activity of the two mutants was observed compared to the native protein thus a correlation of the two mutations and factor’s activity was attempt.|
|Τίτλος πηγής δημοσίευσης: ||Amino Acids 33:3(Sept2007):XXXIX, 10th International Congress on Amino Acids and Proteins (ICAAP), Kallithea, Chalkidiki, Greece, 20 - 25 August, 2007|
|Ηλεκτρονική διεύθυνση περιοδικού (link) : ||http://www.springer.at/amino_acids|
|Εμφανίζεται στις συλλογές:||Άλλα|
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