Purification of a biologically active recombinant glyceraldehyde 3-phosphate dehydrogenase from Candida albicans

We report here the purification of a functionally active recombinant glycer aldehyde 3-phosphate dehydrogenase (GAPDH) from Candida albicans. The GAPDH protein encoded by the TDH1 gene was obtained as a glutathione S-transfera se fusion protein by expression in the vector pGEX-4T-3, and purified by af finity chromatography and thrombin digestion. The purified protein displays GAPDH enzymatic activity (42 mu mol NADH min(-1) mg(-1)) as well as the la minin and fibronectin binding activities previously described. In addition, the recombinant GAPDH is covalently modified by NAD linkage; this modifica tion is stimulated by nitric oxide and probably involves a sulfhydryl group (cysteine) residue since it is inhibited by Hg2+ and cysteine. (C) 1999 Pu blished by Elsevier Science B.V. All rights reserved.