Expression in E. coli and purification of recombinant fragments of wild type and mutant human prion protein

A. Corsaro, S. Thellung, C. Russo, V. Villa, S. Arena, M. C. D'Adamo, D. Paludi, D. Rossi Principe, G. Damonte, U. Benatti, A. Aceto, F. Tagliavini, G. Schettini, T. Florio

Research output: Contribution to journalArticle

Abstract

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, PrPC) into an altered isoform (the prion scrapie, PrPSc), 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 PrPSc. 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 α-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.

Original languageEnglish
Pages (from-to)55-63
Number of pages9
JournalNeurochemistry International
Volume41
Issue number1
DOIs
Publication statusPublished - 2002

Fingerprint

Escherichia coli
Peptides
Transferases
Neurodegenerative Diseases
Protein Isoforms
Immobilized Proteins
Scrapie
Proteins
Prion Diseases
Acrylamide
Prions
Brain
Circular Dichroism
Recombinant Proteins
Immunoblotting
Amyloid
Thrombin
Disulfides
Sepharose
Gel Chromatography

Keywords

  • Glutathione S-transferase-fusion protein
  • Purification
  • Recombinant prion protein
  • Refolding

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology
  • Cellular and Molecular Neuroscience

Cite this

Expression in E. coli and purification of recombinant fragments of wild type and mutant human prion protein. / Corsaro, A.; Thellung, S.; Russo, C.; Villa, V.; Arena, S.; D'Adamo, M. C.; Paludi, D.; Rossi Principe, D.; Damonte, G.; Benatti, U.; Aceto, A.; Tagliavini, F.; Schettini, G.; Florio, T.

In: Neurochemistry International, Vol. 41, No. 1, 2002, p. 55-63.

Research output: Contribution to journalArticle

Corsaro, A, Thellung, S, Russo, C, Villa, V, Arena, S, D'Adamo, MC, Paludi, D, Rossi Principe, D, Damonte, G, Benatti, U, Aceto, A, Tagliavini, F, Schettini, G & Florio, T 2002, 'Expression in E. coli and purification of recombinant fragments of wild type and mutant human prion protein', Neurochemistry International, vol. 41, no. 1, pp. 55-63. https://doi.org/10.1016/S0197-0186(01)00137-1
Corsaro, A. ; Thellung, S. ; Russo, C. ; Villa, V. ; Arena, S. ; D'Adamo, M. C. ; Paludi, D. ; Rossi Principe, D. ; Damonte, G. ; Benatti, U. ; Aceto, A. ; Tagliavini, F. ; Schettini, G. ; Florio, T. / Expression in E. coli and purification of recombinant fragments of wild type and mutant human prion protein. In: Neurochemistry International. 2002 ; Vol. 41, No. 1. pp. 55-63.
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AU - Corsaro, A.

AU - Thellung, S.

AU - Russo, C.

AU - Villa, V.

AU - Arena, S.

AU - D'Adamo, M. C.

AU - Paludi, D.

AU - Rossi Principe, D.

AU - Damonte, G.

AU - Benatti, U.

AU - Aceto, A.

AU - Tagliavini, F.

AU - Schettini, G.

AU - Florio, T.

PY - 2002

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