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A novel copper-binding protein with characteristics of a metallothionein from a clinical isolate of Candida albicans

Department of Biotechnology, Faculty of Technology, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei, Tokyo 184-8588, Japan¹

Author for correspondence: Hideaki Matsuoka. Tel: +81 423 88 7029. Fax: +81 423 87 1503. e-mail: bio-cell{at}cc.tuat.ac.jp

It is known that clinical isolates of Candida albicans exhibit a high level of resistance to copper salts, although the molecular basis of this resistance is not clear. To investigate this, a novel copper-binding protein was purified from a clinical isolate of C. albicans. The protein was extracted from yeast cells after an induction period of 10 h in a copper-containing suspension medium. It was further purified by size-exclusion chromatography, ultrafiltration and reverse-phase HPLC. All protein fractions were analysed for their protein and copper contents. The copper/protein ratio increased steadily throughout the purification process; the most highly purified fraction showed a 210-fold increase compared to the whole-cell extract, with a recovery of 0·03%. The molecular mass of the protein was 10000 Da and a reconstitution study using the purified apoprotein suggested that the equivalent extent of Cu(I) binding was approximately 14 mol eq. The amino-terminal segment of the copper-binding protein revealed three Cys-Xaa-Cys motifs, which is typical of a metallothionein (MT), and showed significant homology with mammalian MTs with respect to the positions of the cysteine residues. This is the first report of the isolation of a copper-binding protein from C. albicans.

Keywords: Candida albicans, copper-binding protein, protein purification and characterization, amino-terminal sequence, metallothionein

Abbreviations: CBB, Coomassie brilliant blue; MT, metallothionein; SDTC, sodium diethyldithiocarbamate

^a Present address: Natural Products Research Institute, Seoul National University, 28 Yungun-dong, Jongro-ku, Seoul 110-460, Korea.

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