NR AOWU
AU Mertz,K.D.; Brunner,E.; Moos,R.; Aguzzi,A.
TI Isolation of Macromolecular Complexes of PrP and Cofactors Using the Tandem Affinity Purification (TAP) Method
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - BR-19
PT Konferenz-Poster
AB The physiological function of the highly conserved prion protein (PrPc) and the mechanisms that control PrPc conversion into the pathological form PrPsc are still unclear. The aim of this study is to identify and characterize the unknown factors that interact differentially with the isoforms of the prion protein, PrPc and PrPsc, using new proteomics tools and tagging strategies. Since the prion protein is N-terminally processed during its conversion into PrPsc, we have C-terminally tagged the prion protein with a novel tandem affinity purification (TAP) tag. The TAP tag allows rapid purification of protein complexes expressed at their natural level under native conditions without prior knowledge of the complex composition, activity or function. Combined with mass spectrometry, the TAP strategy allows for the identification of proteins interacting with a given target protein. The TAP method was initially developed in yeast but can be adapted to various organisms. The open-reading frame of the TAP-tagged prion protein was subcloned into a eucaryotic expression vector and also into the "half-genomic construct" pPrPHG, a PrP-encoding vector containing 6 kb of 5' and 2.2 kb of 3' flanking sequence. This construct was expressed transgenically in Prnp0/0 mice. Protein extracts were purified from several lines of these transgenic mice, and from eukaryotic cell lines that transiently express PrPTAP under the control of the CMV promoter, using the TAP technique. The purified, PrPTAP-associated proteins are being analyzed by mass spectrometry.
AD Kirsten D. Mertz, Erich Brunner, Rita Moos, Adriano Aguzzi, Institute of Neuropathology, Zürich, Switzerland
SP englisch
PO Deutschland