title: Non-reducing alkaline solubilization and rapid on-column refolding of recombinant prion protein.
abstract: Mature prion protein (PrP) is a 208-residue polypeptide that contains a single disulfide bond. We report an alternative method to purify recombinant mouse PrP produced in Escherichia coli. Bacterial inclusion bodies were solubilized in a buffer containing 2 M urea at pH 12.5. The solubilized protein was rapidly purified on a nickel affinity column without a chaotrope gradient, followed by ion-exchange chromatography. The yield and purity of PrP produced by this alternative approach was similar to that obtained using a conventional solubilization and on-column refolding protocol. Recombinant PrP produced using the non-reducing purification protocol is properly folded, as determined by circular dichroism, and a competent substrate for amyloid fibril formation, as determined by Thoflavin-T dye binding assays. In summary, this report describes a rapid method for producing properly folded recombinant PrP without reducing agents or a chaotrope gradient.
authors.affiliation: Departments of Biochemistry, Hanover, New Hampshire 03755, USA.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Walsh
authors.forename: Geoffrey P
authors.initials: GP
authors.lastname: Noble
authors.forename: Justin R
authors.initials: JR
authors.lastname: Piro
authors.forename: Surachai
authors.initials: S
authors.lastname: Supattapone
mesh_terms: D000818:Animals; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D002942:Circular Dichroism / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D000072002:Prion Proteins; D011328:Prions / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
chemical_list: D000072002:Prion Proteins; D011328:Prions; C512657:Prnp protein, mouse; D011994:Recombinant Proteins