Purification and partial characterization of the xanthine-uric acid transporter (UapA) of Aspergillus nidulans.
UapA, the uric acid-xanthine permease from the filamentous ascomycete Aspergillus nidulans, is one of the most thoroughly characterized purine/H(+) transporters in eukaryotes. Detailed studies have addressed its regulation of expression, at both the transcriptional and post-translational levels, in response to physiological and developmental signals. An extensive kinetic profile towards a plethora of purines and mutational analyses have established models on how UapA recognizes the purine ring and revealed specific amino acid residues involved in proper folding, topogenesis, function and specificity. The present work describes for the first time the purification of the UapA transporter of A. nidulans through overexpression via the strong, ethanol-inducible, glucose-repressible, alcA promoter. Purification, almost to homogeneity, was achieved by Ni(2+) affinity chromatography using a functional His-tagged UapA protein version. It is subsequently shown, by Circular Dichroism (CD) spectroscopy, that the purified protein is structured with a high alpha-helical content, as expected from the in silico predictions. The result of this work opens the way for further, analytical and biochemical studies on UapA at the protein level.
Lemuh
Njimoh Dieudonné
ND
Laboratory of Microbiology, Faculty of Biology, Department of Botany, University of Athens, Zografou Campus, Athens 15784, Greece.
Diallinas
George
G
Frillingos
Stathis
S
Mermelekas
George
G
Karagouni
Amalia D
AD
Hatzinikolaou
Dimitris G
DG
D001233:Aspergillus nidulans / Q000201:enzymology*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D000431:Ethanol / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D015151:Immunoblotting; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013057:Spectrum Analysis
D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; C083852:UAPA protein, Aspergillus nidulans; D000431:Ethanol; D005947:Glucose