High yield purification and physico-chemical properties of full-length recombinant allelic variants of sheep prion protein linked to scrapie susceptibility.
Sheep susceptibility to scrapie is governed by polymorphisms at two major sites, codons 136 and 171, of the prp gene. To get more insight into the prion protein (PrP) sequence-linked basis of differential scrapie susceptibility, a high yield one-step method for the purification (over 99% final purity) of the full-length recombinant sheep PrP was developed, based on the affinity of the conserved octapeptide repeats for transition-metal cations. Thermal and chemical denaturation experiments and limited proteolysis studies were performed on the natural variants (A136R171, V136Q171 and A136Q171) and a recombinant PrP mutated at position 136 (V136R171). Results revealed the influence of mutations in positions 136 and 171 on the folding thermodynamic parameters and on the conformation of the C-terminal domain. Together, our results show that the VQ cellular protein linked to higher scrapie susceptibility is intrinsically more compact and/or stable than the resistance-linked AR counterpart. This might lead to a lower in vivo clearance rate of VQ and a consequently higher probability of occurrence of pathological events.
Rezaei
H
H
Institut National de la Recherche Agronomique (INRA) Unité 806/EA2703, Muséum National d'Histoire Naturelle, Institut de Biologie Physico-Chimique, Paris, France.
Marc
D
D
Choiset
Y
Y
Takahashi
M
M
Hui Bon Hoa
G
G
Haertlé
T
T
Grosclaude
J
J
Debey
P
P
D000483:Alleles; D000818:Animals; D015153:Blotting, Western; D002942:Circular Dichroism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D020022:Genetic Predisposition to Disease; D006863:Hydrogen-Ion Concentration; D008957:Models, Genetic; D016296:Mutagenesis; D011110:Polymorphism, Genetic; D011328:Prions / Q000235:genetics* / Q000302:isolation & purification*; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012608:Scrapie / Q000235:genetics / Q000378:metabolism*; D012756:Sheep; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors
D018076:DNA, Complementary; D011328:Prions; D011994:Recombinant Proteins; D019286:Endopeptidase K