title: Purification and characterization of the extracellular aspartyl proteinase of Candida albicans: removal of extraneous proteins and cell wall mannoprotein and evidence for lack of glycosylation.
abstract: Aspartyl proteinase (AP) is an extracellular enzyme of Candida albicans implicated as a pathogenic factor. Previous reports on the purification and characterization of AP suggested that a single DEAE-Sephadex chromatographic step was sufficient for the removal of extraneous proteins and that the final product was glycosylated. We purified AP using a chromatographic series consisting of DEAE-Sephadex A25, Sephadex G75 and rechromatography on DEAE-Sephadex A25. Use of DEAE-Sephadex alone did not remove extraneous proteins and removed little contaminating mannoprotein (MP). The addition of a Sephadex G75 column to the purification scheme removed the majority of contaminating MP and proteins. The final DEAE-Sephadex A25 chromatographic step resulted in (a) removal of detectable extraneous proteins, (b) removal of immunologically detectable MP by dot blot and Western blot enzyme immunoassay, (c) loss of periodic acid-silver stain positivity, and (d) a high AP yield (1295 U l-1) and specific activity (1749 U mg-1). We conclude that a single DEAE-Sephadex A25 purification step is insufficient to remove extraneous proteins and MP, which could interfere with the production of AP-specific antibodies and the dissection of moieties responsible for immune reactivity. Reports of periodic acid-Schiff or anthrone positivity of AP preparations may reflect the presence of extraneous MP, which can be removed by the chromatographic series we describe.
authors.affiliation: Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333.
authors.forename: C J
authors.initials: CJ
authors.lastname: Morrison
authors.forename: S F
authors.initials: SF
authors.lastname: Hurst
authors.forename: S L
authors.initials: SL
authors.lastname: Bragg
authors.forename: R J
authors.initials: RJ
authors.lastname: Kuykendall
authors.forename: H
authors.initials: H
authors.lastname: Diaz
authors.forename: D W
authors.initials: DW
authors.lastname: McLaughlin
authors.forename: E
authors.initials: E
authors.lastname: Reiss
mesh_terms: D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000302:isolation & purification*; D002176:Candida albicans / Q000201:enzymology* / Q000302:isolation & purification / Q000472:pathogenicity; D002177:Candidiasis / Q000175:diagnosis; D002473:Cell Wall / Q000737:chemistry; D002852:Chromatography, Ion Exchange / Q000379:methods; D005110:Extracellular Space / Q000201:enzymology; D005656:Fungal Proteins / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D008562:Membrane Glycoproteins / Q000302:isolation & purification
chemical_list: D005656:Fungal Proteins; D008562:Membrane Glycoproteins; C025363:mannoproteins; D016282:Aspartic Acid Endopeptidases