title: Construction of FLAG tagging vectors for Candida albicans.
abstract: We have constructed three new vectors for Candida albicans (pFLAG-Act1, pFLAG-Mal2, and pFLAG-Met3). The proteins can be expressed as C-terminal FLAG-tagged proteins under the control of different promoters (ACT1, MAL2, and MET3). To confirm the protein expression, we used the Renilla reniformis luciferase and the drug efflux pump Cdr1p of Candida albicans as reporters. The luciferase protein expressed by the MET3 promoter was found to have the strongest activity of the three promoters when cultured in a methionine-depleted synthetic medium. Cdr1p was expressed as a C-terminal FLAG-tagged protein using either these vectors or PCR-mediated integration. The fluconazole resistance was increased by the Cdr1p expression in a CDR1 homozygous disruptant. The expressed proteins were detected by Western blotting using the anti-FLAG antibody. We also constructed a Cdr1p-FLAG expressing strain, in which we directly tagged Cdr1p with FLAG on the genome loci, using a PCR-based integrative marker cassette that was amplified using the pFLAG vector. We then confirmed the protein expression by Western blotting. Thus, these new vectors are useful as C. albicans genetic tools.
authors.affiliation: Department of Bioactive Molecules, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Umeyama
authors.forename: Yuki
authors.initials: Y
authors.lastname: Nagai
authors.forename: Masakazu
authors.initials: M
authors.lastname: Niimi
authors.forename: Yoshimasa
authors.initials: Y
authors.lastname: Uehara
mesh_terms: D000199:Actins / Q000235:genetics; D001483:Base Sequence; D002176:Candida albicans / Q000235:genetics*; D004271:DNA, Fungal / Q000032:analysis; D000939:Epitopes / Q000235:genetics; D015725:Fluconazole; D005656:Fungal Proteins; D017930:Genes, Reporter; D005822:Genetic Vectors; D008156:Luciferases / Q000235:genetics; D026901:Membrane Transport Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics*; D011401:Promoter Regions, Genetic; D029702:Schizosaccharomyces pombe Proteins; D016324:Sequence Tagged Sites; D014170:Transformation, Genetic
chemical_list: C404682:ACT1 protein, Candida albicans; D000199:Actins; C094693:CDR1 protein, Candida albicans; D004271:DNA, Fungal; D000939:Epitopes; D005656:Fungal Proteins; C102110:Mal2 protein, S pombe; D026901:Membrane Transport Proteins; D009687:Nuclear Proteins; D009842:Oligopeptides; D010455:Peptides; D029702:Schizosaccharomyces pombe Proteins; D015725:Fluconazole; C085706:FLAG peptide; D008156:Luciferases