title: Prion protein from Xenopus laevis: overexpression in Escherichia coli of the His-tagged protein and production of polyclonal antibodies.
abstract: Prion protein (PrP) and PrP-related proteins have been identified in reptiles, amphibians, and fishes by means of cDNA cloning, genome database searching and comparative genomics. However, no studies have been reported so far on the expression of PrP at the protein level in those animals. This report presents a procedure to obtain and purify recombinant PrP from Xenopus laevis expressed in Escherichia coli as a fusion protein in which mature PrP (residues 21-194) is linked to a 35-amino acid N-terminal extension containing a hexahistidine stretch. The protein was used to raise and purify by affinity chromatography anti-Xenopus PrP polyclonal antibodies which were suitable to detect the presence of PrP in Xenopus brain by Western blot. This is the first report of a positive identification of PrP in amphibian at the protein level. Anti-Xenopus PrP antibodies do not cross react with PrP from different sources (human, bovine, sheep, and turtle). Similarly, Xenopus PrP do not react with anti-turtle PrP(143-248) antibodies.
authors.affiliation: D.I.P.A.V. Section of Biochemistry, University of Milano, Via Celoria 10, 20133 Milano, Italy.
authors.forename: Lara
authors.initials: L
authors.lastname: Pagliato
authors.forename: Armando
authors.initials: A
authors.lastname: Negri
authors.forename: Simona
authors.initials: S
authors.lastname: Nonnis
authors.forename: Francesca
authors.initials: F
authors.lastname: Taverna
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Sangiorgio
authors.forename: Severino
authors.initials: S
authors.lastname: Ronchi
authors.forename: Gabriella
authors.initials: G
authors.lastname: Tedeschi
mesh_terms: D029845:Amphibian Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D015153:Blotting, Western / Q000379:methods; D001923:Brain Chemistry / Q000276:immunology; D002846:Chromatography, Affinity / Q000379:methods; D004926:Escherichia coli; D011328:Prions / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D013045:Species Specificity; D014426:Turtles / Q000276:immunology; D014982:Xenopus laevis
chemical_list: D029845:Amphibian Proteins; D000906:Antibodies; D011328:Prions; D011993:Recombinant Fusion Proteins