A novel tandem affinity purification strategy for the efficient isolation and characterisation of native protein complexes.
Isolation and dissection of native multiprotein complexes is a central theme in functional genomics. The development of the tandem affinity purification (TAP) tag has enabled an efficient and large-scale purification of native protein complexes. However, the TAP tag features a size of 21 kDa and requires time consuming cleavage. By combining a tandem Strep-tag II with a FLAG-tag we were able to reduce the size of the TAP (SF-TAP) tag to 4.6 kDa. Both moieties have a medium affinity and avidity to their immobilised binding partners. This allows the elution of SF-tagged proteins under native conditions using desthiobiotin in the first step and the FLAG octapeptide in the second step. The SF-TAP protocol represents an efficient, fast and straightforward purification of protein complexes from mammalian cells within 2.5 h. The power of this novel method is demonstrated by the purification of Raf associated protein complexes from HEK293 cells and subsequent analysis of their protein interaction network by dissection of interaction patterns from the Raf binding partners MEK1 and 14-3-3.
Gloeckner
Christian Johannes
CJ
Institute of Human Genetics, GSF-National Research Center for Environment and Health, Neuherberg, Germany.
Boldt
Karsten
K
Schumacher
Annette
A
Roepman
Ronald
R
Ueffing
Marius
M
D048948:14-3-3 Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D001710:Biotin / Q000031:analogs & derivatives / Q000737:chemistry; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D048369:MAP Kinase Kinase 1 / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000302:isolation & purification*; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D048493:Proto-Oncogene Proteins B-raf / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014162:Transfection
D048948:14-3-3 Proteins; C099517:Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; D046912:Multiprotein Complexes; D009842:Oligopeptides; D010455:Peptides; C482983:YWHAE protein, human; D001710:Biotin; C004749:desthiobiotin; C085706:FLAG peptide; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human