Proteomic identification, nucleotide sequence, heterologous expression and immunological reactivity of the triosephosphate isomerase of Paracoccidioides brasiliensis.
An antigen of Paracoccidioides brasiliensis (Pb) was gel isolated and characterized. Endoproteinase Lys-C-digested peptides of the purified protein, which presented a molecular mass of 29 kDa and pI of 5.8, were subjected to sequence analysis of their amino acids. Searches at databases comparing the sequence of amino acids from the three peptides of the native protein revealed strong homology to triosephosphate isomerase (TPI: E.C. 5.3.1.1) from several sources. The complete cDNA and gene encoding PbTPI were obtained and both contained an open reading frame predicted to encode a 249-amino acid protein that presented all the peptides characterized in the native PbTPI. The Pbtpi gene contained six exons interrupted by five introns. Analysis performed with the deduced PbTPI suggested its usefulness in providing phylogenetic relatedness, as well as evidencing the correlation between the phylogeny provided by the deduced protein and intron positions in the cognate genes. The immunological reactivity of PbTPI was examined. The complete coding cDNA of PbTPI was overexpressed in an Escherichia coli host to produce high levels of recombinant fusion protein with glutathione S-transferase (GST) that had been purified by affinity chromatography. The purified recombinant TPI was recognized by sera of patients with confirmed paracoccidioidomycosis and not by sera of healthy individuals. Thus, recombinant PbTPI can be a valuable addition to the still small arsenal of P. brasiliensis immunoreactive proteins, which could be tested for incorporation into assays for serodiagnosis of the disease.
Laboratório de Biologia Molecular, ICBII, ICB2, Campus 2, UFG, Universidade Federal de Goiás, 74001-970, Goiânia, Goiás, Brazil.
Luiz A
LA
Pereira
Maristela
M
Pereira
Maria S S
MS
Felipe
Rosely M
RM
Zancopé-Oliveira
Célia M de Almeida
CM
Soares
D000595:Amino Acid Sequence; D000908:Antibodies, Fungal / Q000097:blood; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010228:Paracoccidioides / Q000201:enzymology* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D010229:Paracoccidioidomycosis / Q000382:microbiology; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D040901:Proteomics; D016415:Sequence Alignment; D014305:Triose-Phosphate Isomerase / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000908:Antibodies, Fungal; D004271:DNA, Fungal; D014305:Triose-Phosphate Isomerase