| resuspended in 100 ml buffer G (6 M GdmCl, 10 mM Tris-HCl, 100 mM Na2PO4, 10 mM reduced glutathione, pH 8.0), and further processed as outlined in Fig. 1. After sonication and centrifugation, the soluble protein fraction was added to 30 ml of nickel-nitrilotriacetic acid (NTA) agarose resin (Qiagen) and stirred for 10 min. The resin was poured into a column and washed with 120 ml buffer G, and to the immobilized histidine tail-containing fusion protein we applied a 200 ml gradient of buffer G to buffer B (10 mM Tris-HCl, 100 mM Na2PO4, pH 8.0). Protein impurities devoid of histidine tails were removed from the agarose resin with 75 ml of 50 mM imidazole in buffer B. The polypeptides hPrP(23?31), hPrP(81?31), and hPrP(90?31) were eluted with buffer E (10 mM Tris, 100 mM Na2PO4, 500 mM imidazole, pH 5.8), while the fragment hPrP(121?31) was eluted with buffer B containing 150 mM imidazole. After washing the resin with 30 ml of buffer G, oxidative refolding and imidazole elution were repeated to obtain a second batch of soluble prion protein. Histidine tail-fused PrP was dialysed against buffer D (10 mM Na2PO4, pH 5.8), and afterwards against water. The histidine tail was removed from the prion protein using thrombin (Sigma; 0.1 units/ml). The cleaving reaction was carried out at RT for 1 h in buffer C (5 mM Tris-HCl buffer, pH 8.5). The protease was removed from the prion protein fragment hPrP(121?31) by loading onto a DE 52 column (10 ml resin; Whatman) equilibrated in buffer C, followed by elution with a 200 ml gradient of 0?00 mM NaCl. The polypeptides hPrP(23?31), hPrP(81?31) and hPrP(90?31) were loaded onto a CM 52 resin (Whatman) equilibrated with buffer I (10 mM Na2PO4, pH 6.5), and eluted with a 200 ml gradient of 0?00 mM NaCl. The cleaved histidine tail was removed by dialysis against water was using a Spectrapor-membrane (MW 6000?000) |