NR AOYP
AU Sakudo,A.; Hamaishi,M.; Hosokawa-Kanai,T.; Tuchiya,K.; Nishimura,T.; Saeki,K.; Matsumoto,Y.; Ueda,S.; Onodera,T.
TI Purification and characterization of a soluble cellular isoform of prion protein produced by baculovirus expression system
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - BR-73
PT Konferenz-Poster
AB The function of cellular isoform of prion protein (PrPc) has not been fully elucidated. It is partly due to the difficulty in obtaining an adequate quantity of PrPc for biochemical and structural characterizations. Here, a method for expression and purification of a soluble form of histidine (HIS)-tagged murine prion protein (bacMuPrP), which lacks the entire C-terminal cleavage and glycosyl phosphatidyl inositol (GPI) addition site, has been developed using a recombinant baculovirus expression system and purification with Ni-NTA agarose affinity chromatography. In mammalian sources, PrPc is attached to the cell membrane by a GPI anchor. However, in our system, bacMuPrP was secreted into the media, enabling its easy purification in abundance. Although a previous report indicates hundreds of normal hamster brains yielded only a few micrograms of protein, the yield of bacMuPrP after purification in our system achieved an order of 1 microgram per 10 ml of culture medium. Tunicamycin treatment revealed non-glycosylated proteins were secreted into the media, suggesting that glycosylation is not necessary for bacMuPrP secretion. Density-gradient sedimentation analysis demonstrated a sedimentation coefficient of secretory bacMuPrP as 2.3 S, indicating a monomeric form. Although affinity-purified prion protein (PrP) from mouse brain or recombinant PrP produced by Escherichia coli and refolded in the presence of copper has been reported to display superoxide dismutase (SOD) activity, bacMuPrP did not show SOD activity. These results suggest that bacMuPrP has a different biochemical and biophysical characterization from mammalian and bacterial-derived PrP. Furthermore, this simple expression system may provide an adequate source for structural and biochemical analysis of PrP.
AD Akikazu Sakudo, Michiko Hamaishi, Takuya Nishimura, Keiichi Saeki, Yoshitsugu Matsumoto, Takashi Onodera, Dept. of Mol. Immunol., University of Tokyo, Japan; Tomoko Hosokawa-Kanai, Kotaro Tuchiya, Susumu Ueda, Nippon Institute for Biological Science, Japan
SP englisch
PO Deutschland