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Biotechnology and Applied Biochemistry (1999) 29, (223–227) (Printed in Great Britain)

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Generation of a highly immunogenic recombinant enolase ofthe human opportunistic pathogen Candida albicans

Silvia Sandini*, Roberta Melchionna†, SilviaArancia*, Maria Jesus Gomez* and Roberto La Valle*¹

*Laboratorio di Batteriologia e Micologia Medica, IstitutoSuperiore di Sanita, Viale Regina Elena 299, 00161 Rome, Italy,and †Dipartimento di Istologia ed Embriologia Medica,Universita La Sapienza, Via Scarpa 14, 00161 Rome, Italy

Abbreviations used: CMI response, cell-mediated immune response; GST, glutathione S-transferase; His₆-tagged enolase, Candida albicans enolase fused to a hexahistidine affinity domain; IPTG, isopropyl b--thiogalactoside; LB, Luria–Bertani; PBMC, peripheral blood mononuclear cells.

¹ To whom correspondence should be addressed.

Enolase, a 46 kDa glycolytic enzyme, is an immunodominant antigen of Candida albicans, an important human opportunistic pathogen. The full-length coding sequence of C. albicans enolase gene was subcloned into the prokaryotic expression vector pDS56/RBSII,His₆/E^- under the control of an inducible promoter to produce a His₆-tagged enolase. The recombinant protein was purified to homogeneity by one-step nickel-chelate affinity chromatography. It was recognized by a monoclonal antibody specific for C. albicans enolase, as well as by anti-enolase antibodies present in human sera (IgG). The recombinant protein promptly elicited antibody in mice and detected immune responses in normal human subjects that were comparable to those generated by the native C. albicans enolase. Thus this new recombinant enolase constitutes a valuable reagent for studying the possible role of this protein in anti-Candida immune response.

Received 13 November 1998/9 February 1999; accepted 12 February 1999

Portland Press Ltd © 1999