Expression in E. coli and purification of recombinant fragments of wild type and mutant human prion protein.
Prion diseases are fatal neurodegenerative disorders of the CNS of men and animals, characterized by spongiform degeneration of the CNS, astrogliosis and deposition of amyloid into the brain. The conversion of a cellular glycoprotein (the prion protein, PrP(C)) into an altered isoform (the prion scrapie, PrP(Sc)), which accumulates within the brain tissue by virtue of its resistance to the intracellular catabolism, is currently believed to represent the etiologic agent responsible for these diseases. Synthetic or recombinant polypeptides are commonly used to elucidate the mechanism of proteins involved in neurodegenerative diseases. Here we describe a procedure, which allows the synthesis and purification in its native folding, of the human prion protein fragment 90-231, corresponding to the protease resistant core of PrP(Sc). We synthesized the polypeptides 90-231 of both the wild type and the E200K mutant isoforms of PrP. Using a gluthatione S-transferase (GST) fusion protein approach, milligram amounts of polypeptides were obtained after expression in E. coli. The recovery of the purified fusion protein was monitored following the evaluation of the GST activity. The PrP fragment was released from the fusion protein immobilized on a glutathione-coupled agarose resin by direct cleavage with thrombin. The recombinant protein was identified by comassie stained acrylamide gel and by immunoblotting employing a monoclonal anti-PrP antibody. The peptide purified by gel filtration chromatography showed mainly an alpha-helix structure, as analysed by circular dichroism (CD) and an intact disulfide bridge. The same procedure was also successfully employed to synthesize and purify the E200K mutant PrP fragment.
Corsaro
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Pharmacology and Neuroscience, National Institute for Cancer Research (IST), c/o Advanced Biotechnology Center (CBA), Genoa, Italy.
Thellung
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Russo
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Villa
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Arena
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D'Adamo
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MC
Paludi
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Rossi Principe
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Damonte
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Benatti
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Aceto
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Tagliavini
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Schettini
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Florio
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D001483:Base Sequence; D015153:Blotting, Western; D002853:Chromatography, Liquid; D002942:Circular Dichroism; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D013058:Mass Spectrometry; D011328:Prions / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence
D017931:DNA Primers; D011328:Prions; D011994:Recombinant Proteins