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Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans
Published on: January 8, 2015
Absence of superoxide dismutase activity in a soluble cellular isoform of prion protein produced by baculovirus
Akikazu Sakudo1, Michiko Hamaishi, Tomoko Hosokawa-Kanai
1Department of Molecular Immunology, School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Abstract:
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.
Insights
A novel method expresses soluble histidine-tagged murine prion protein (bacMuPrP) using a baculovirus system. This secreted protein is easily purified and shows distinct characteristics from mammalian and bacterial PrP, offering a new source for research.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Expression
Background:
- Mammalian Prion Protein (PrP(C)) is typically membrane-bound via a glycosyl phosphatidyl inositol (GPI) anchor.
- Existing methods for recombinant prion protein (PrP) production have limitations in solubility and purification.
- Understanding PrP structure and function requires readily available, soluble protein forms.
Purpose of the Study:
- To develop a method for expressing and purifying a soluble, secreted form of histidine-tagged murine prion protein (bacMuPrP).
- To characterize the biochemical and biophysical properties of the expressed bacMuPrP.
- To assess the suitability of bacMuPrP as a source for further PrP research.
Main Methods:
- Utilized a recombinant baculovirus expression system for bacMuPrP production.
- Employed Ni-NTA agarose affinity chromatography for protein purification.
- Conducted indirect immunofluorescence, immunoblot analysis, tunicamycin treatment, and density-gradient sedimentation.
Main Results:
- Successfully expressed and purified soluble bacMuPrP, which was secreted into the media.
- Localized bacMuPrP to the perinuclear endoplasmic reticulum and the media, not the cell membrane.
- Demonstrated that glycosylation is not essential for bacMuPrP secretion and that it exists as a monomer (2.3 S).
- Found that bacMuPrP lacks superoxide dismutase (SOD) activity, differing from other PrP forms.
Conclusions:
- The baculovirus expression system provides an efficient method for producing soluble, secreted bacMuPrP.
- bacMuPrP exhibits unique biochemical and biophysical characteristics compared to mammalian and bacterial PrP.
- This system offers a valuable and accessible source for structural, functional, and biochemical studies of prion protein.
