YEAST-ENHANCED GREEN FLUORESCENT PROTEIN (YEGFP) - A REPORTER OF GENE-EXPRESSION IN CANDIDA-ALBICANS

The green fluorescent protein (GFP) of Aequorea victoria has been deve loped here as a reporter for gene expression and protein localization in Candida albicans. When wild-type (wt) GFP was expressed in C. albic ans, it was not possible to detect fluorescence or a translation produ ct for the wt protein. Since this was probably due in part to the pres ence of the non-canonical CTG serine codon in the Aequorea sequence, t his codon was changed to the leucine codon TTG. C. albicans cells expr essing this construct contained GFP mRNA but were non-fluorescent and contained no detectable translation product. Hence a codon-optimized G FP gene was constructed in which all of the 239 amino acids are encode d by optimal codons for C. albicans. In this gene were also incorporat ed two previously identified mutations in the chromophore that increas e GFP fluorescence. C. albicans cells expressing this yeast-enhanced G FP gene (yEGFP3) are fluorescent and contain GFP protein. yEGFP3 can b e used as a versatile reporter of gene expression in C. albicans and S accharomyces cerevisiae and the optimized GFP described here should ha ve broad applications in these and other fungal species.