title: Purification of a biologically active recombinant glyceraldehyde 3-phosphate dehydrogenase from Candida albicans.
abstract: We report here the purification of a functionally active recombinant glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Candida albicans. The GAPDH protein encoded by the TDH1 gene was obtained as a glutathione S-transferase fusion protein by expression in the vector pGEX-4T-3, and purified by affinity chromatography and thrombin digestion. The purified protein displays GAPDH enzymatic activity (42 micromol NADH min(-1) mg(-1)) as well as the laminin and fibronectin binding activities previously described. In addition, the recombinant GAPDH is covalently modified by NAD linkage; this modification is stimulated by nitric oxide and probably involves a sulfhydryl group (cysteine) residue since it is inhibited by Hg(2+) and cysteine.
authors.affiliation: Departamento de Microbiología y Ecología, Facultad de Farmacia, Universitat de València, Avda. Vicent Andrés Estellés, s/n, 46100 Burjassot, Valencia, Spain.
authors.forename: E
authors.initials: E
authors.lastname: Villamón
authors.forename: D
authors.initials: D
authors.lastname: Gozalbo
authors.forename: J P
authors.initials: JP
authors.lastname: Martínez
authors.forename: M L
authors.initials: ML
authors.lastname: Gil
mesh_terms: D002176:Candida albicans / Q000201:enzymology* / Q000235:genetics; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005982:Glutathione Transferase / Q000096:biosynthesis; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005982:Glutathione Transferase; D013917:Thrombin