Expression and purification of recPrP.

(A) Truncated ovine recPrP was expressed in, and purified from, E. coli Rosetta (DE3) competent cells (Merck). The process was monitored by analysis of fractions on 12% SDS-PAGE gels, with proteins visualised by Coomassie blue: M, molecular mass marker (units in kDa); U, sample from uninduced cells prior to induction with IPTG; I, sample from induced cells; S, sample from supernatant after clarification by centrifugation; B₁, sample taken from buffer while bedding resin in column; B₂, sample from flow-through while bedding resin; D, sample from denaturing step with guanidine; R₁, sample from gradient refolding; R₂, sample form isocratic refolding; E₇ and E₈, eluted fractions containing recPrP, as evidenced by an approximate 17 kDa protein band corresponding to monomeric truncated ovine recPrP; X, sample from resin cleaning step in which any remaining protein was denatured and stripped from the resin. (B) Samples from eluted fractions were assessed for purity by silver staining: E₁-E₉, samples from gradient elution with imidazole; F₁, final dialysed recPrP stock; F₂, final recPrP stock passed through 100 kDa MWCO spin filter. (C) Elution of recPrP was monitored by UV. The fraction corresponding to the middle 50% of the elution peak (A_{280 nm} > 0.4 AU) (Fraction E7/E8, green) was pooled to yield 27 mg recPrP. Original, uncropped and minimally-adjusted gel images are provided in another supplementary information file (S1_raw_images).

(TIF)