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

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.