Vectors and gene targeting modules for tandem affinity purification in Schizosaccharomyces pombe

We describe the construction of tagging cassettes and plasmids for tandem a ffinity purification (TAP) of proteins in Schizosaccharomyces pombe, The ta gging 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-resista nt cells. The amino-terminal tagging vectors allow for the regulated expres sion of proteins. St. pombe Cdc2p was chosen to test these new affinity tag s. Several known binding proteins co-purified with both Cdc2p-CTAP and N-TA P-Cdc2p, indicating the usefulness of these tags for the rapid purification of stable protein complexes from Sz. pombe. Copyright (C) 2001 John Wiley & Sons, Ltd.