Expression and purification of active human internal His6-tagged l-glutamine: d-Fructose-6P amidotransferase I

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 His6-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 KmFru-6P=0.98mM and KmGln=0.84mM 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 Kd of 5 μM for Fru-6P.