Tandem affinity purification of the Candida albicans septin protein complex.

A novel vector was constructed to enable the integrative marking of individual genes and the affinity purification of interacting molecules within protein complexes from Candida albicans using a tandem 6 x histidine and FLAG epitope tag. The system was verified by purifying the C. albicans septin complex (a self-associating complex ...
of cytoskeletal proteins) from both yeast and hyphal cells. One-step affinity purification was insufficient for purification of the protein complex, whereas tandem affinity purification (TAP) gave an extensively purified protein complex with a very low background. Electrophoretic and mass spectrometry analysis showed that the affinity-purified C. albicans septin complex, which comprises predominantly CaCdc3p, CaCdc10p, CaCdc11p, CaCdc12p and CaSep7p, was not affected by cell morphology. The purified septin complex appeared to have a stoichiometry of 2 CaCdc3p, 1-2 CaCdc10p, 1 CaCdc11p, 2 CaCdc12p and < or = 1 CaSep7p. The successful application of TAP to the purification and analysis of the C. albicans septin complex indicates that this technology will have much wider application to proteomic studies of this pathogenic fungus.
Mesh Terms:
Amino Acid Sequence, Base Sequence, Candida albicans, Chromatography, Affinity, Cytoskeletal Proteins, Electrophoresis, Gel, Two-Dimensional, Epitopes, Fungal Proteins, Histidine, Molecular Sequence Data, Peptide Mapping, Peptides, Proteome, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Yeast
Date: Sep. 01, 2004
Download Curated Data For This Publication
155912
Switch View:
  • Interactions 25