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 t o macrophages and is a major target antigen of immune recognition in a cquired resistance to the fungus. In past studies, WI-1 has been purif ied by extracting the protein from the yeast cell wall, which yields m icrogram quantities for biological assays. We report a strategy for ge nerating and purifying the secreted form of WI-1 in quantity. Yeasts o f B. dermatitidis ATCC strain 60636 cultured in HMM medium were found to secrete 10 mu g/ml of WI-1 into a supernate relatively free of othe r medium and yeast components. Using a two-step method of ion exchange and hydrophobic interaction chromatography, we achieved a 7.1-fold pu rification of WI-1, Purified WI-1 was sequenced at the N-terminus whic h revealed that the secreted protein exists in two different forms. In functional assays, purified WI-1 also retained its adhesivity for hum an macrophages, and its antigenicity in binding anti-WI-1 antibodies a nd stimulating T-cells to proliferate, but it lost some capacity to el icit 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 relationshi p between three-dimensional structure and activity of the molecule. (C ) 1997 Academic Press.