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

• Published in: Fungal genetics and biology Vol. 20; no. 2; pp. 125 - 131
• Main Authors: McEwen, J G, Ortiz, B L, García, A M, Florez, A M, Botero, S, Restrepo, A
• Format: Journal Article
• Language: English
• Published: United States 01-06-1996
• Subjects: Amino Acid Sequence; Antigens, Fungal - chemistry; Antigens, Fungal - genetics; Antigens, Fungal - isolation & purification; Base Sequence; Cloning, Molecular; DNA, Complementary - genetics; Escherichia coli - genetics; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - isolation & purification; Genes, Fungal - genetics; Molecular Sequence Data; Molecular Weight; Paracoccidioides - genetics; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - isolation & purification; Restriction Mapping; Sequence Analysis, DNA
• ISSN: 1087-1845
• DOI: 10.1006/fgbi.1996.0027

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 immuno-screened clones from the library, 2 were positive (clones 2 and 3). The former was not characterized further. The latter has a 1-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.