کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1926555 1536466 2008 10 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Large-scale expression, purification, and characterization of an engineered prostacyclin-synthesizing enzyme with therapeutic potential
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Large-scale expression, purification, and characterization of an engineered prostacyclin-synthesizing enzyme with therapeutic potential
چکیده انگلیسی
Recently, we reported that a novel hybrid enzyme (TriCat enzyme), engineered by linking human cyclooxygenase-2 (COX-2) with prostacyclin (PGI2) synthase (PGIS) together through a transmembrane domain, was able to directly integrate the triple catalytic (TripCat) functions of COX-2 and PGIS and effectively convert arachidonic acid (AA) into the vascular protector, PGI2 [K.H. Ruan, H. Deng, S.P. So, Biochemistry 45 (2006) 14003-14011]. In order to confirm the important biological activity and evaluate its therapeutic potential, it is critical to characterize the properties of the enzyme using the purified protein. The TriCat enzyme cDNA was subcloned into a baculovirus vector and its protein was expressed in Sf-9 cells in large-scale with a high-yield (∼4% of the total membrane protein), as confirmed by Western blot and protein staining. The Sf-9 cells' membrane fraction, rich in TriCat enzyme, exhibited strong TriCat functions (Km = 3 μM and Kcat = 100 molecules/min) for the TriCat enzyme and was 3-folds faster in converting AA to PGI2 than the combination of the individual COX-2 and PGIS. Another superiority of the TriCat enzyme is its dual effect on platelet aggregation: it completely inhibited platelet aggregation at the low concentration of 2 μg/ml and then displayed the ability to reverse the initially aggregated platelets to their non-aggregated state. Furthermore, multiple substrate-binding sites were confirmed in the single protein by high-resolution NMR spectroscopy, using partially purified TriCat enzyme. These studies have clearly demonstrated that the isolated TriCat enzyme protein functions in the selective biosynthesis of the vascular protector, PGI2, and revealed its potential for anti-thrombosis therapeutics.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Archives of Biochemistry and Biophysics - Volume 480, Issue 1, 1 December 2008, Pages 41-50
نویسندگان
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