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 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.
Fisher
K L
KL
University of Wisconsin Medical School, Department of Medical Microbiology and Immunology, Madison, WI 53706-1532, USA.
Woods
J P
JP
D000946:Antigens, Fungal / Q000235:genetics / Q000378:metabolism*; D017077:Culture Media, Conditioned / Q000378:metabolism; D004274:DNA, Recombinant / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D006639:Histidine / Q000235:genetics; D006658:Histoplasma / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000378:metabolism*
D000946:Antigens, Fungal; D017077:Culture Media, Conditioned; D004274:DNA, Recombinant; D011994:Recombinant Proteins; D006639:Histidine; D001617:beta-Glucosidase