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.