1 - Laboratório de Biologia Molecular, ICB II, UFG, Goiânia, Goiás, Brasil.

2 - Laboratório de Biologia Molecular, ICB, UnB, Brasília, DF, Brasil.

Keywords: Paracoccidioides brasiliensis; Triosephosphate isomerase; Gene cloning and structural analysis; Recombinant protein.

The homodimeric enzyme triosephosphate isomerase (TPI) is widely distributed in nature, converting glyceraldehyde-3-phosphate to dehydroxyacetone phosphate, an important step in the glycolytic pathway. The antigenic proprieties of this enzyme its has been evidencieted in some microorganisms. Studies with S. japonicum TPI have indicated that immunization of mice with the protein can promote reduction in liver pathology. Other studies with the T. brucei TPI purpose the design of new potential inhibitors for this enzyme. The P. brasiliensis TPI was recognized in Western blot assays by PCM patients antibodies. In our work we characterized the complete cDNA and genomic sequences of PbTPI. The heterologous expression and purification of PbTPI was been performed, and the purified recombinant protein was obtained.

A yeast cDNA library was constructed into EcoRI and XhoI sites of Lambda Zap II (Stratagene/LaJolla/CA/USA). The screening of this library was performed using the 949 bp fragment radiolabelled with [a -³²P] dCTP. The in vivo excision of pBluescript phagemids (Stratagene) in Escherichia coli XL1 Blue MRF´ was performed. The nucleotide sequence was determined on both strands.

The complete genomic sequence encoding TPI was obtained by PCR amplification of the total DNA of P. brasiliensis. Primers were constructed based on the cDNA sequence. It was obtained a 1350 bp PCR product by using sense primer S1 and the C-terminal At2 as the antisense primer.

The phylogenetic relationships of PbTPI were generated with 17 selected sequences from organisms of diverse taxon presenting identity to PbTPI and with all the fungi TPI complete protein and genomic sequences available at databases. The entire compared amino acid sequences were visualized using the TreeView software. A phylogenetic tree was constructed by multiple sequence alignments using the Clustal X program. Trees were constructed by using the Neighbor-Joining method.

Oligonucleotide primers were designed to amplify the 750 bp complete cDNA of Pbtpi. The nucleotide sequences of the sense and antisense primers contained engineered EcoRI and XhoI restriction sites, respectively. The 750 bp PCR product was cloned into the EcoRI/XhoI sites of pGEX-4T-3 (Amersham, Biosciences) to yield the pGEX-4T-3-TPI construct. The recombinant plasmid was used to transform the E. coli XL1 Blue competent cells using the heat shock method. 0.1mM IPTG was used to induce the recombinant protein expression. The recombinant TPI was expressed in the soluble form by the bacteria and the protein was purified by affinity chromatography under nondenaturing conditions. The purity and size of the recombinant protein were evaluated by running the purified molecule on a 12% SDS-PAGE followed by Coomassie blue staining.

The P. brasiliensis nucleotide sequences and the deduced amino acid sequences have been submitted to the GenBank database under accession numbers: AY250089 (cDNA) and AY037936 (genomic). Sequences analyses were performed by using the BLAST program, the PROSITE and Pfam databases.

The characterized TPI of P. brasiliensis, isolate Pb01, is reactive to serum of PCM patients. This data were the first criteria used for the posterior characterization of TPI encoding sequences, and the protein heterologous expression. In order to isolate the complete gene and cDNA encoding TPI of P. brasiliensis we obtained initially a PCR product comprehending a fragment of the tpi gene, the 949 bp PCR product was used as a probe to screen a P. brasiliensis cDNA library. The entire cDNA consisted of 1080 bp with a 750 bp open reading frame coding for a protein with 249 amino acid residues. The deduced amino acid sequence of PbTPI shows high homology with other TPI sequences described in database. The predicted molecular mass was of to 29 kDa, in agreement to the SDS-PAGE analysis of the nature TPI (29 kDa). The structural alignment of this sequence evidencieted putative 8 a -helices and 8 b -sheets regions in accordance with descriptions of other TPIs in database.

The complete genomic sequence was obtained by PCR and was compared to the cDNA. Comparative analysis of the cDNA and genomic sequences of PbTPI confirmed the intron positions and sizes. The five introns found in Pbtpi gene its present in other fungal TPIs, with many variations of the intron positions in other sequences analyzed. The patterns obtained in Southern blot analysis suggest that the PbTPI gene its present in only one copy in the P. brasiliensis genome.

PbTPI ORF (750bp) was amplified by PCR with Eco RI/Xho I adictional sites, and cloned in pGEX-4-T3^® expression vector. The bacterial induction product was analyzed by SDS-PAGE. A pattern with 56kDa its in a overexpression. The fusion protein was cleaved by addition of thrombin protease and the purification of recombinant PbTPI was realized.

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