Prion protein from Xenopus laevis: overexpression in Escherichia coli of the His-tagged protein and production of polyclonal antibodies.
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.
Pagliato
Lara
L
D.I.P.A.V. Section of Biochemistry, University of Milano, Via Celoria 10, 20133 Milano, Italy.
Negri
Armando
A
Nonnis
Simona
S
Taverna
Francesca
F
Sangiorgio
Lorenzo
L
Ronchi
Severino
S
Tedeschi
Gabriella
G
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
D029845:Amphibian Proteins; D000906:Antibodies; D011328:Prions; D011993:Recombinant Fusion Proteins