The application of tandem-affinity purification to Candida albicans.
Tandem-affinity purification (TAP) tagging systems, developed by the research group of Bertrand Seraphin and others, are a means of isolating physiologically relevant protein and protein-nucleic acid complexes. Where complete (or nearly complete) genome sequence data are available for the organism from which the complexes are isolated, their components can be readily identified using mass spectrometry. The most widely used TAP-tag consists of a proximal calmodulin-binding peptide (CBP) and a distal repeated protein A IgG-binding domain with a cleavage site for the tobacco etch virus (TEV) protease positioned between this and the CBP. This tag is expressed as a co-translational fusion to the protein of interest. Purification is achieved under mild conditions through sequential affinity chromatography on IgG (eluting by proteolytic cleavage with TEV protease) and calmodulin (eluting by removal of Ca(2+) ions required for the interaction) resins. The approach has been hugely successful for categorizing the interactome of Saccharomyces cerevisiae. Here, we present vectors for carrying out TAP-tagging in Candida albicans and a protocol for purification of complexes containing TAP-tagged proteins.
Blackwell
Chris
C
Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne, UK.
Brown
Jeremy D
JD
D002176:Candida albicans / Q000737:chemistry* / Q000235:genetics; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005656:Fungal Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D005821:Genetic Techniques
D005656:Fungal Proteins