Determination of beta-glucosidase enzymatic function of the Histoplasma capsulatum H antigen using a native expression system

The Histoplasma capsulatum H antigen is a major secreted glycoprotein of th is pathogenic fungus that is a target of humoral and cell-mediated host res ponses. Its predicted protein sequence displays homology to beta-glucosidas es of other organisms, but a recombinant antigen expressed in a prokaryotic system showed no enzymatic activity. We expressed a recombinant form of th e protein carrying a carboxyl-terminus oligohistidine tag in the native fun gal 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 tr ansformant carrying the modified gene under the control of its native promo ter. The purified protein from the native expression system showed beta-glu cosidase enzymatic activity in substrate gels and quantitative microplate a ssays. 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-tran slational modification necessary for structural and functional integrity. ( C) 2000 Elsevier Science B.V. All rights reserved.