Purification in quantity of the secreted form of WI-1: a major adhesin on Blastomyces dermatitidis yeasts.
WI-1, a 120-kDa adhesin on Blastomyces dermatitidis, binds the yeast to macrophages and is a major target antigen of immune recognition in acquired resistance to the fungus. In past studies, WI-1 has been purified by extracting the protein from the yeast cell wall, which yields microgram quantities for biological assays. We report a strategy for generating and purifying the secreted form of WI-1 in quantity. Yeasts of B. dermatitidis ATCC strain 60636 cultured in HMM medium were found to secrete 10 microg/ml of WI-1 into a supernate relatively free of other medium and yeast components. Using a two-step method of ion exchange and hydrophobic interaction chromatography, we achieved a 7.1-fold purification of WI-1. Purified WI-1 was sequenced at the N-terminus which revealed that the secreted protein exists in two different forms. In functional assays, purified WI-1 also retained its adhesivity for human macrophages, and its antigenicity in binding anti-WI-1 antibodies and stimulating T-cells to proliferate, but it lost some capacity to elicit delayed-type hypersensitivity in mice. These findings advance our understanding of the WI-1 adhesin/antigen and our ability to express and purify WI-1 in quantity and will permit a study of the relationship between three-dimensional structure and activity of the molecule.
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Comprehensive Cancer Center, University of Wisconsin Medical School, Madison, Wisconsin 53792, USA.
Brandhorst
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Klein
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D000595:Amino Acid Sequence; D000818:Animals; D000908:Antibodies, Fungal; D000946:Antigens, Fungal / Q000276:immunology / Q000302:isolation & purification*; D001758:Blastomyces / Q000737:chemistry*; D015815:Cell Adhesion Molecules / Q000276:immunology / Q000302:isolation & purification*; D002473:Cell Wall / Q000737:chemistry; D003470:Culture Media; D005656:Fungal Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D006023:Glycoproteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D006968:Hypersensitivity, Delayed; D008213:Lymphocyte Activation; D051379:Mice; D008969:Molecular Sequence Data; D017421:Sequence Analysis; D013601:T-Lymphocytes / Q000187:drug effects; D015003:Yeasts / Q000737:chemistry*
D000908:Antibodies, Fungal; D000946:Antigens, Fungal; D015815:Cell Adhesion Molecules; D003470:Culture Media; D005656:Fungal Proteins; D006023:Glycoproteins