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
Lubos
L
Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.
Spirek
Mario
M
Novatchkova
Maria
M
Chen
Zhiming
Z
Rumpf
Cornelia
C
Lugmayr
Wolfgang
W
Mechtler
Karl
K
Ammerer
Gustav
G
Csaszar
Edina
E
Gregan
Juraj
J
D002846:Chromatography, Affinity / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012568:Schizosaccharomyces / Q000737:chemistry* / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000302:isolation & purification*
D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins