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The application of tandem-affinity purification to Candida albicans

Lookup NU author(s): Christopher Blackwell, Dr Jeremy Brown


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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 Ca2+ 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. © 2009 Humana Press.

Publication metadata

Author(s): Blackwell C; Brown J

Editor(s): Ronald L. Cihlar and Richard A. Calderon

Publication type: Book Chapter

Publication status: Published

Book Title: Candida albicans: Methods and protocols

Year: 2009

Volume: 499

Pages: 133-148

Print publication date: 01/01/2009

Series Title: Methods in Molecular Biology

Publisher: Humana Press

Place Published: New York


DOI: 10.1007/978-1-60327-151-6_13

PubMed id: 19152045

Library holdings: Search Newcastle University Library for this item

ISBN: 9781588297600