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  • Title: Expression and purification of active human internal His(6)-tagged L-glutamine: D-Fructose-6P amidotransferase I.
    Author: Richez C, Boetzel J, Floquet N, Koteshwar K, Stevens J, Badet B, Badet-Denisot MA.
    Journal: Protein Expr Purif; 2007 Jul; 54(1):45-53. PubMed ID: 17379537.
    Abstract:
    Human L-glutamine: D-fructose-6-phosphate amidotransferase (Gfat1), a recognized target in type 2 diabetes complications, was expressed in Sf9 insect cells with an internal His(6)-tag and purified to homogenity. Two different microplate assays that quantify, respectively D-glucosamine-6-phosphate and L-glutamate were used to analyze the enzyme kinetic properties. The recombinant human L-glutamine: D-fructose-6-phosphate amidotransferase isoform 1 exhibits Michaelis parameters K(m)(Fru-6P)=0.98 mM and K(m)(Gln)=0.84 mM which are similar to the values reported for the same enzyme from different sources. The stimulation of hydrolysis of the alternate substrate L-glutamine para-nitroanilide by D-fructose-6P (Fru-6P) afforded a K(d) of 5 microM for Fru-6P.
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