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    Biochem Biophys Res Commun. 2006 Jun 9;344(3):1008-16. Epub 2006 Apr 19.

    Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase.

    Brizio C, Galluccio M, Wait R, Torchetti EM, Bafunno V, Accardi R, Gianazza E, Indiveri C, Barile M.

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    Dipartimento di Biochimica e Biologia Molecolare Ernesto Quagliariello, Università degli Studi di Bari, Via Orabona 4, I-70126 Bari, Italy.

    Abstract

    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 63kDa 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 60kDa 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 MgCl(2), as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8+/-1.3nmol of FAD synthesized/min/mg protein and exhibited a K(M) value for FMN of 1.5+/-0.3microM. 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.

    PMID:
    16643857
    [PubMed - indexed for MEDLINE]

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