The Effects of Novel Mutations in A1 Domain of Human Coagulation Factor VIII on Its Secretion Level in Cultured Mammalian Cells
نویسندگان: ثبت نشده
چکیده مقاله:
Inefficient secretion of the human coagulation factor (hFVIII) in mammalian expression systems is one ofthe main causes of the hFVIII low expression level, attributed to its interaction with a chaperone known asBiP/GRP78. In order to improve secretion efficiency of the hFVIII, based on the higher secretion level of theporcine FVIII and analysis of the hFVIII A110 region, that inhibits its secretion, function of three novel Bdomain deleted hFVIII mutant including; two singlemutants (Leu299Phe and Phe309Thr) and a doublemutant (Tyr323His/Lys325Arg) were examined in three mammalian cell lines (HEK-293T, COS, CHO) for the hFVIII secretion efficiency. The double-mutant construct displayed the highest hFVIII expression level,about seven-fold as much the base-line. The doublemutant hFVIII was biologically active and its inactivationpatterns by EDTA and heat was similar to that of the non-mutant hFVIII. Semi-quantitative RT-PCRresults showed the highest mRNA level for the doublemutant hFVIII. Both of the mutated residues in the double-mutant are located in a hydrophobic heptamer (320MEAYVKV326) that seems to be involved in Bipbinding activity. None of the L299F and F309T hFVIII mutants exhibited improved secretion. This result hasprovided convincing evidence for the increasing effect of the double-mutant on the hFVIII secretion and transcription efficiencies.
منابع مشابه
Expression of Biologically Active Recombinant B-Domain-Deleted Human Factor VIII in Mammalian Cells
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عنوان ژورنال
دوره 8 شماره 3
صفحات 139- 149
تاریخ انتشار 2010-07-01
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