title: Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli.
abstract: The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)6-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni(2+)-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner.
authors.affiliation: Virologie et Immunologie Moléculaires, INRA.
authors.forename: Qiaojing
authors.initials: Q
authors.lastname: Li
authors.affiliation: Virologie et Immunologie Moléculaires, INRA.
authors.forename: Charles-Adrien
authors.initials: CA
authors.lastname: Richard
authors.affiliation: Virologie et Immunologie Moléculaires, INRA.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Moudjou
authors.affiliation: Virologie et Immunologie Moléculaires, INRA; jasmina.vidic@jouy.inra.fr.
authors.forename: Jasmina
authors.initials: J
authors.lastname: Vidic
mesh_terms: D000595:Amino Acid Sequence; D000682:Amyloid / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D000818:Animals; D002850:Chromatography, Gel / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D058851:GPI-Linked Proteins; D006639:Histidine / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D009842:Oligopeptides / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D017510:Protein Folding; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014508:Urea / Q000737:chemistry
chemical_list: D000682:Amyloid; D058851:GPI-Linked Proteins; C471213:His-His-His-His-His-His; D009419:Nerve Tissue Proteins; D009842:Oligopeptides; D011994:Recombinant Proteins; C523856:Sprn protein, mouse; D006639:Histidine; D014508:Urea