XXXX -binding protein in Candida albicans: antibody development and cellular localization by electron immunocytochemistry.

@entity1 @entity172 @entity2931 @entity53 @entity5132 @entity375 @entity32799
@entity5132 , the most common fungal pathogen of @entity1 , possesses an @entity2931 ( @entity172 )-binding protein ( @entity32799 ) that binds oestrogens with high affinity and specificity. The gene that encodes the @entity32799 (CaEBP1) has been cloned and sequenced and shown to be structurally related to the old yellow enzyme from @entity375 . Here, we report the purification and the subcellular localization of the @entity32799 from @entity5132 . Using ion-exchange chromatography and an @entity53 affinity column, the @entity32799 was purified from a strain of @entity5132 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 @entity32799 , depleted the @entity2931 -binding activity from @entity5132 cell extracts. Western blot analysis showed that the antisera specifically recognized the @entity32799 from C. albicans. The antibodies also recognized the protein when the cloned CaEBP1 gene was expressed in @entity375 and did not cross react with @entity375 proteins. Using electron microscopy and antigen detection by immunogold staining, the @entity32799 appeared to be primarily associated with vacuoles. However, when overexpressed in @entity375 , the @entity32799 was found diffusely throughout the cell. In conclusion, the @entity32799 has been purified from C. albicans and antibodies generated against the protein were used to demonstrate that @entity32799 is found associated with vacuoles in @entity5132 .
