Production, purification and oxidative folding of the mouse recombinant prion protein.
The method leading to overexpression of the full-length mouse recombinant prion protein (mrPrP 23-231) in the cytoplasm of E. coli as a his-PrP fusion protein and its effective purification using affinity chromatography is described. A typical yield of the method was 8-10 mg his-mrPrP per L of the bacterial culture. The purity of purified protein was > 95 %. The purified his-mrPrP was converted to a soluble form and its folding to alpha-helical and beta-sheet conformations was studied. The properties of differently folded mrPrP were determined by measuring their circular dichroism spectra, partial resistance to cleavage by proteinase K and by centrifugation in sucrose gradient.
Institute of Microbiology and Immunology, 1st Faculty of Medicine, Charles University, 128 00 Prague, Czechia.
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Pavlícek
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Bednárová
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Holada
D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D051379:Mice; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011328:Prions / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D010446:Peptide Fragments; D011328:Prions; D011994:Recombinant Proteins; C428437:prion protein (23-231)