title: Oestrogen-binding protein in Candida albicans: antibody development and cellular localization by electron immunocytochemistry.
abstract: Candida albicans, the most common fungal pathogen of humans, possesses an oestrogen (estrogen)-binding protein (EBP) that binds oestrogens with high affinity and specificity. The gene that encodes the EBP (CaEBP1) has been cloned and sequenced and shown to be structurally related to the old yellow enzyme from Saccharomyces cerevisiae. Here, we report the purification and the subcellular localization of the EBP from C. albicans. Using ion-exchange chromatography and an oestradiol affinity column, the EBP was purified from a strain of C. albicans (strain 422) which was selected because it constitutively expressed elevated levels of the binding protein. The purified protein displayed a subunit molecular mass of approximately 46 kDa when examined by denaturing gel electrophoresis, which is consistent with the size estimated from the sequence of the cloned CaEBP1 gene. An immunoaffinity column, prepared using a polyclonal antisera generated against EBP, depleted the oestrogen-binding activity from C. albicans cell extracts. Western blot analysis showed that the antisera specifically recognized the EBP from C. albicans. The antibodies also recognized the protein when the cloned CaEBP1 gene was expressed in S. cerevisiae and did not cross react with S. cerevisiae proteins. Using electron microscopy and antigen detection by immunogold staining, the EBP appeared to be primarily associated with vacuoles. However, when overexpressed in S. cerevisiae, the EBP was found diffusely throughout the cell. In conclusion, the EBP has been purified from C. albicans and antibodies generated against the protein were used to demonstrate that EBP is found associated with vacuoles in C. albicans.
authors.affiliation: Stanford University School of Medicine, Division of Endocrinology, Ca 94305, USA.
authors.forename: X
authors.initials: X
authors.lastname: Zhao
authors.forename: P J
authors.initials: PJ
authors.lastname: Malloy
authors.forename: C M
authors.initials: CM
authors.lastname: Ardies
authors.forename: D
authors.initials: D
authors.lastname: Feldman
mesh_terms: D015153:Blotting, Western; D002176:Candida albicans / Q000254:growth & development / Q000648:ultrastructure*; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D002451:Cell Compartmentation; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004958:Estradiol / Q000378:metabolism; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000648:ultrastructure*; D016253:Microscopy, Immunoelectron; D011960:Receptors, Estrogen; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000648:ultrastructure; D014617:Vacuoles / Q000648:ultrastructure*
chemical_list: D002352:Carrier Proteins; D011960:Receptors, Estrogen; D011994:Recombinant Proteins; C010140:estrophilin; D004958:Estradiol