Absence of superoxide dismutase activity in a soluble cellular isoform of prion protein produced by baculovirus expression system.
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, PrP(C) 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. Indirect immunofluorescence studies and immunoblot analysis localized not in cell membrane but in the perinuclear endoplasmic reticulum region in cells and is secreted into the media. 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 PrP from mouse brain or recombinant prion protein (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, functional, and biochemical analyses of PrP.
Sakudo
Akikazu
A
Department of Molecular Immunology, School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Hamaishi
Michiko
M
Hosokawa-Kanai
Tomoko
T
Tuchiya
Kotaro
K
Nishimura
Takuya
T
Saeki
Keiichi
K
Matsumoto
Yoshitsugu
Y
Ueda
Susumu
S
Onodera
Takashi
T
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D051379:Mice; D018621:PrPC Proteins / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics; D013482:Superoxide Dismutase / Q000378:metabolism*
D018621:PrPC Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D013482:Superoxide Dismutase