Molecular cloning, characterization and expression of the heat shock protein 60 gene from the human pathogenic fungus Paracoccidioides brasiliensis

A gene encoding the heat shock protein (HSP) 60 from Paracoccidioides brasi liensis (Pb) was cloned and characterized. The, hsp60 gene is composed of t hree exons divided by two introns. Structural analysis of the promoter dete cted canonical sequences characteristic of regulatory regions from eukaryot ic genes. The deduced amino acid sequence of the Pb hsp60 gene and the resp ective cloned cDNA consists of 592 residues highly homologous to other fung al HSP60 proteins. The hsp60 gene is present as a single copy in the genome , as shown by Southern blot analysis. The HSP60 protein was isolated from P b yeast cellular extracts. N-terminal amino acid sequencing of HSP60 confir med that the cloned hsp60 gene correlated to the predicted protein in Pb. H SP60 expression appeared to be regulated during form transition in Pb, as d ifferent levels of expression were detected in in vitro labeling of cells a nd northern blot analysis. The complete coding region of Job hsp60 was fuse d with plasmid pGEX-4T-3 and expressed in Escherichia coli as a glutathione S-transferase-tagged recombinant protein. The protein reacted with a mouse monoclonal antibody raised to a human recombinant HSP60. Western immunoblo t experiments demonstrated that the recombinant protein and the native HSP6 0 were recognized by sera from humans with paracoccidioidomycosis (PCM).