title: Improved Tandem Affinity Purification Tag and Methods for Isolation of Proteins and Protein Complexes fromSchizosaccharomyces pombe.
abstract: The tandem affinity purification (TAP) method uses an epitope that contains two different affinity purification tags separated by a site-specific protease site to isolate a protein rapidly and easily. Proteins purified via the TAP tag are eluted under mild conditions, allowing them to be used for structural and biochemical analyses. The original TAP tag contains a calmodulin-binding peptide and the IgG-binding domain from protein A separated by a tobacco etch virus (TEV) protease cleavage site. After capturing the Protein A epitope on an IgG resin, bound proteins are released by incubation with the TEV protease and then isolated on a calmodulin matrix in the presence of calcium; elution from this resin is achieved by chelating calcium with EGTA. However, because the robustness of the calmodulin-binding step in this procedure is highly variable, we replaced the calmodulin-binding peptide with three copies of the FLAG epitope, (3× FLAG)-TEV-Protein A, which can be isolated using an anti-FLAG resin. Elution from this matrix is achieved in the presence of an excess of a 3× FLAG peptide. In addition to allowing proteins to be released under mild conditions, elution by the 3× FLAG peptide adds an extra layer of specificity to the TAP procedure, because it liberates only FLAG-tagged proteins.
authors.affiliation: Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037.
authors.forename: Nicola
authors.initials: N
authors.lastname: Zilio
authors.affiliation: Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037 nboddy@scripps.edu.
authors.forename: Michael N
authors.initials: MN
authors.lastname: Boddy
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D012568:Schizosaccharomyces / Q000737:chemistry*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins