An improved strategy for tandem affinity purification-tagging of Schizosaccharomyces pombe genes.
Tandem affinity purification (TAP) is a method that allows rapid purification of native protein complexes. We developed an improved technique to fuse the fission yeast genes with a TAP tag. Our technique is based on tagging constructs that contain regions homologous to the target gene cloned into vectors carrying a TAP tag. We used this technique to design strategies for TAP-tagging of predicted Schizosaccharomyces pombe genes (http://mendel.imp.ac.at/Pombe_tagging/). To validate the approach, we purified the proteins, which associated with two evolutionarily conserved proteins Swi5 and Sfr1 as well as three protein kinases Ksg1, Orb6 and Sid1.
Cipak L, Spirek M, Novatchkova M, Chen Z, Rumpf C, Lugmayr W, Mechtler K, Ammerer G, Csaszar E, Gregan J