Recombinant full-length murine prion protein, mPrP(23-231): purification and spectroscopic characterization.
The cellular prion protein of the mouse, mPrP(C), consists of 208 amino acids (residues 23-231). It contains a carboxy-terminal domain, mPrP(121-231), which represents an autonomous folding unit with three alpha-helices and a two-stranded antiparallel beta-sheet. We expressed the complete amino acid sequence of the prion protein, mPrP(23-231), in the cytoplasm of Escherichia coli. mPrP(23-231) was solubilized from inclusion bodies by 8 M urea, oxidatively refolded and purified to homogeneity by conventional chromatographic techniques. Comparison of near-UV circular dichroism, fluorescence and one-dimensional 1H-NMR spectra of mPrP(23-231) and mPrP(121-231) shows that the amino-terminal segment 23-120, which includes the five characteristic octapeptide repeats, does not contribute measurably to the manifestation of three-dimensional structure as detected by these techniques, indicating that the residues 121-231 might be the only polypeptide segment of PrP(C) with a defined three-dimensional structure.
Hornemann
S
S
Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, Zürich, Switzerland.
Korth
C
C
Oesch
B
B
Riek
R
R
Wider
G
G
Wüthrich
K
K
Glockshuber
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry; D018621:PrPC Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence
D004220:Disulfides; D010446:Peptide Fragments; D018621:PrPC Proteins; D011993:Recombinant Fusion Proteins