Analysis of Secreted Aspartic Proteinases From Candida Albicans: Purification and Characterization of Individual Sap1, Sap2 and Sap3 Isoenzymes

Microbiology. 1997 Feb;143 ( Pt 2):349-56. doi: 10.1099/00221287-143-2-349.


The recently discovered secreted aspartic proteinase multi-gene (SAP) family in Candida albicans has complicated assessment of proteolytic activity as a factor in the onset and development of Candida infections. Differential expression of the SAP genes under various conditions, as well as possible variation in the properties of the individual isoenzymes, have consequences for immunological detection, for targeted drug design and possibly for pathogenicity. It is therefore important to be able to monitor Sap isoenzyme profiles in different strains of C. albicans cultures, and to know the biochemical properties of each isoenzyme. We have employed a simple purification protocol based on strong anion exchange chromatography for the direct analysis of C. albicans Sap isoenzymes from culture filtrates, as well as recovery of individual Sap1, Sap2 and Sap3 products. In the case of Sap1, this involved development of an overexpression system using the pEMBLyex4 vector transformed into Saccharomyces cerevisiae. The C. albicans strains ATCC 10231 and 10261 were shown to produce different ratios of Sap2 and Sap3 under the same conditions. Analysis of all three purified proteins by gel electrophoresis, immunoblotting and proteinase assays which were designed to evaluate pH dependence, thermal stability and substrate specificity revealed similar but distinct properties for each isoenzyme. Although Sap3 was shown to be antigenically more similar to Sap2 than was Sap1, it was less similar in terms of thermal stability and activity at low pH, being more stable and more active.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aspartic Acid Endopeptidases / genetics
  • Aspartic Acid Endopeptidases / isolation & purification*
  • Aspartic Acid Endopeptidases / metabolism
  • Candida albicans / enzymology*
  • Chromatography, High Pressure Liquid
  • Chromatography, Ion Exchange
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / isolation & purification
  • DNA-Binding Proteins / metabolism
  • Enzyme Stability
  • Fungal Proteins*
  • Isoenzymes / isolation & purification*
  • Recombinant Proteins / biosynthesis
  • Saccharomyces cerevisiae / genetics
  • Schizosaccharomyces pombe Proteins*


  • DNA-Binding Proteins
  • Fungal Proteins
  • Isoenzymes
  • Recombinant Proteins
  • Sap1 protein, S pombe
  • Schizosaccharomyces pombe Proteins
  • Aspartic Acid Endopeptidases
  • SAP2 protein, Candida
  • SAP3 protein, Candida albicans