Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase
- Dipartimento di Biochimica e Biologia Molecolare 'Ernesto Quagliariello', Universita degli Studi di Bari, Via Orabona 4, I-70126 Bari (Italy)
- Dipartimento di Biologia Cellulare, Universita della Calabria, Via P. Bucci 4c, I-87036 Arcavacata di Rende (Italy)
- Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College London, 1 Aspenlea Road, Hammersmith, London W6 8LH (United Kingdom)
- Infections and Cancer Biology Group, International Agency for Research on Cancer, World Health Organization, 150 Cours Albert Thomas, 69372 Lyon (France)
- Gruppo di Studio per la Proteomica e la Struttura delle Proteine, Dipartimento di Scienze Farmacologiche, Universita degli Studi di Milano, Via G. Balzaretti 9, I-20133 Milan (Italy)
- Dipartimento di Biochimica e Biologia Molecolare 'Ernesto Quagliariello', Universita degli Studi di Bari, Via Orabona 4, I-70126 Bari (Italy) and Istituto di Biomembrane e Bioenergetica, C.N.R., Via Amendola 165/A, I-70126 Bari (Italy)
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 MgCl{sub 2}, 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 K {sub M} value for FMN of 1.5 {+-} 0.3 {mu}M. This is First report on characterization of human FADS, and First cloning and over-expression of FADS from an organism higher than yeast.
- OSTI ID:
- 20798996
- Journal Information:
- Biochemical and Biophysical Research Communications, Vol. 344, Issue 3; Other Information: DOI: 10.1016/j.bbrc.2006.04.003; PII: S0006-291X(06)00801-1; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA); ISSN 0006-291X
- Country of Publication:
- United States
- Language:
- English
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