Molecular cloning and expression of a 70-kilodalton heat shock protein of Candida albicans.
By screening an expression library of the yeast form of Candida albicans with a serum directed against whole fungal cells, a cDNA (2,325 bp) encoding a stress protein of C. albicans was cloned and sequenced. The cloned sequence (CaRLV130) identified a single open reading frame with a length of 1,968 bp coding for a protein containing 656 amino acid residues (70 kDa). The deduced amino acid sequence was 84% similar to the sequence of the Saccharomyces cerevisiae SSA1 gene, which encodes one member of the 70-kDa heat shock protein (Hsp70) family. The relevant gene (C. albicans HSP70 gene [CaHSP70]) was localized on the highest-M(r) (R1; approximately 3.8 Mb) chromosome of C. albicans as determined by pulse-field electrophoresis. CaHSP70 was expressed after heat shock, as demonstrated by Northern (RNA) blotting and reverse transcriptase-PCR with specific pairs of oligonucleotide sequences and gene probes. A recombinant protein was obtained in Escherichia coli after cloning of the full coding sequence into the BamHI site of the pDS56/RBSII6xhisE- plasmid and purification by nickel chelate affinity chromatography. The recombinant protein (6xhis-CaHsp70) was efficiently recognized in immunoblots by a monoclonal antibody directed against a common epitope of eukaryotic Hsp70 proteins, as well as by sera from normal human subjects. Moreover, immune mouse sera against the purified recombinant protein recognized native, heat-inducible constituents with sizes of around 70 kDa in whole-cell protein extracts of C. albicans. Overall, our data demonstrate that CaHSP70 encodes one member of a family of proteins (Hsp70) which usually represent highly conserved immunodominant antigens of infectious agents.
La Valle
R
R
Department of Bacteriology and Medical Mycology, Istituto Superiore di Sanità, Rome, Italy.
Bromuro
C
C
Ranucci
L
L
Muller
H M
HM
Crisanti
A
A
Cassone
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002176:Candida albicans / Q000235:genetics*; D002177:Candidiasis / Q000276:immunology; D003001:Cloning, Molecular; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011817:Rabbits
D006360:Heat-Shock Proteins