This study describes a comprehensive purification and characterization of aspartyl proteinase (AP), an extracellular enzyme from Candida albicans implicated in pathogenesis. Previous purification methods using a single DEAE-Sephadex step were insufficient to remove contaminating cell wall mannoprotein (MP) and extraneous proteins. The researchers developed a multi-step chromatographic approach combining DEAE-Sephadex A25, Sephadex G75, and rechromatography on DEAE-Sephadex A25 to achieve high-purity AP with specific activity of 1749 U/mg and good yield. The final preparation contained less than 0.06% contaminating MP and was devoid of immunologically detectable MP by dot blot and Western blot analysis. Contrary to previous reports suggesting AP is glycosylated, periodic acid-silver staining detected no carbohydrate on the purified enzyme, and growth with tunicamycin (a glycosylation inhibitor) had no effect on AP production. The researchers concluded that earlier reports of AP glycosylation likely reflected contaminating MP, not the AP itself. This purified preparation is essential for producing specific anti-AP antibodies and studying the enzyme's pathogenic mechanisms.

Key findings

• A three-step chromatographic purification procedure (DEAE-Sephadex A25, Sephadex G75, and secondary DEAE-Sephadex A25) is necessary to remove extraneous proteins and cell wall mannoprotein, with a single DEAE-Sephadex step being insufficient
• The purified AP preparation contained less than 0.06% contaminating mannoprotein and achieved high specific activity of 1749 U/mg with good recovery
• Purified AP lacks detectable glycosylation, contrary to previous reports; earlier claims of AP glycosylation were likely due to contaminating mannoprotein
• Tunicamycin treatment had no effect on AP production or molecular mass, further supporting that AP is not glycosylated