title: Molecular cloning, nucleotide sequencing, and characterization of a 27-kDa antigenic protein from Paracoccidioides brasiliensis.
abstract: 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.
authors.affiliation: Seccione de Biología Molecular, Corporación para Investigaciones Biológicas (CIB), Medellin, Colombia. cib@medellin.cetcol.net.co
authors.forename: J G
authors.initials: JG
authors.lastname: McEwen
authors.forename: B L
authors.initials: BL
authors.lastname: Ortiz
authors.forename: A M
authors.initials: AM
authors.lastname: García
authors.forename: A M
authors.initials: AM
authors.lastname: Florez
authors.forename: S
authors.initials: S
authors.lastname: Botero
authors.forename: A
authors.initials: A
authors.lastname: Restrepo
mesh_terms: D000595:Amino Acid Sequence; D000946:Antigens, Fungal / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D005800:Genes, Fungal / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010228:Paracoccidioides / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA
chemical_list: C103540:27 kDa protein, Paracoccidioides brasiliensis; D000946:Antigens, Fungal; D018076:DNA, Complementary; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins