Determination of beta-glucosidase enzymatic function of the histoplasma capsulatum H antigen using a native expression system.
Gene
; 247(1-2): 191-7, 2000 Apr 18.
Article
in En
| MEDLINE
| ID: mdl-10773459
ABSTRACT
The Histoplasma capsulatum H antigen is a major secreted glycoprotein of this pathogenic fungus that is a target of humoral and cell-mediated host responses. Its predicted protein sequence displays homology to beta-glucosidases of other organisms, but a recombinant antigen expressed in a prokaryotic system showed no enzymatic activity. We expressed a recombinant form of the protein carrying a carboxyl-terminus oligohistidine tag in the native fungal background to facilitate proper glycosylation and folding of a product that could then be purified from culture supernatants using nickel affinity chromatography. The recombinant protein was expressed and secreted by a transformant carrying the modified gene under the control of its native promoter. The purified protein from the native expression system showed beta-glucosidase enzymatic activity in substrate gels and quantitative microplate assays. This activity was blocked by glucosidase-specific inhibitors. These results are the first direct demonstration of the function of this protein, and show the utility of expression in a native system to achieve post-translational modification necessary for structural and functional integrity.
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Collection:
01-internacional
Database:
MEDLINE
Main subject:
Beta-Glucosidase
/
Histoplasma
/
Antigens, Fungal
Language:
En
Journal:
Gene
Year:
2000
Document type:
Article
Affiliation country: