Molecular cloning, nucleotide sequencing, and characterization of a 27-kDa antigenic protein from Paracoccidioides brasiliensis

McEwen, J. G., Ortiz, B. L., Garcia, A. M., Florez, A. M., Botero, S., and Restrepo, A. 1996, Molecular cloning, nucleotide sequencing, and characterization of a 27-kDa antigenic protein from Paracoccidioides brasiliensis, Fungal Genetics and Biology 20, 125-131, A gene encoding a 27-kDa antigenic protein from Paracoccidioides brasiliensis was cloned, sequenced, and characterized, A cDNA library of the mycelial phase was produced and packed in Uni-Zap-XR vector, lambda Zap II synthesis kit (Stratagene, La Jolla, CA), The screening of the library was carried out using a pool of sera from paracoccidioidomycosis patients that had proven reactive in serological testing, Among 44,000 immunoscreened clones from the library, 2 were positive (clones 2 and 3), The former was not characterized further, The latter has a l-kb DNA insert with an open reading frame encoding a protein of 259 amino acids with a predicted molecular mass of 28.6 kDa (27 kDa by SDS-PAGE), This protein corresponds to a 25-kDa protein in antigenic preparations of P. brasiliensis as determined by Western blot analysis, Comparison of the transcribed sequence with different gene banks failed to reveal a high degree of homology with other proteins, The cloned DNA fragment was easily expressed in Escherichia coli without the need of induction by isopropyl-beta-D-thiogalactopyranoside. These findings suggest that the gene encodes a P. brasiliensis-specific protein. (C) 1996 Academic Press. Inc.