کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1941096 1050797 2006 9 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase
کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase
چکیده انگلیسی

FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63 kDa on SDS–PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60 kDa on SDS–PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl2, as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8 ± 1.3 nmol of FAD synthesized/min/mg protein and exhibited a KM value for FMN of 1.5 ± 0.3 μM. This is the first report on characterization of human FADS, and the first cloning and over-expression of FADS from an organism higher than yeast.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biochemical and Biophysical Research Communications - Volume 344, Issue 3, 9 June 2006, Pages 1008–1016
نویسندگان
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