Vectors and gene targeting modules for tandem affinity purification in Schizosaccharomyces pombe.
We describe the construction of tagging cassettes and plasmids for tandem affinity purification (TAP) of proteins in Schizosaccharomyces pombe. The tagging cassettes are designed for either carboxy- or amino-terminal tagging of proteins. The carboxyl terminal tags differ in that they contain either two or four repeats of IgG binding units. For tagging endogenous loci, the cassettes contain the kan MX6 module to allow for selection of G418-resistant cells. The amino-terminal tagging vectors allow for the regulated expression of proteins. Sz. pombe Cdc2p was chosen to test these new affinity tags. Several known binding proteins co-purified with both Cdc2p-CTAP and N-TAP-Cdc2p, indicating the usefulness of these tags for the rapid purification of stable protein complexes from Sz. pombe.
Tasto
J J
JJ
Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Carnahan
R H
RH
McDonald
W H
WH
Gould
K L
KL
D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000302:isolation & purification; D004271:DNA, Fungal / Q000737:chemistry / Q000302:isolation & purification; D005656:Fungal Proteins / Q000302:isolation & purification*; D005822:Genetic Vectors / Q000138:chemical synthesis / Q000235:genetics*; D016254:Mutagenesis, Insertional / Q000379:methods; D016133:Polymerase Chain Reaction; D012568:Schizosaccharomyces / Q000737:chemistry*
D004271:DNA, Fungal; D005656:Fungal Proteins; D016203:CDC2 Protein Kinase