PEC2132-1 cells were cultured for 3 h at 37uC in 3 L of LB+Kanamycin (50 mg/ml) medium and expression of His 6 -proHNP1 was induced by addition of 0.5 mM isopropyl b-D-1thiogalactopyranoside (IPTG) for an additional 3 h. Cells were harvested by centrifugation and lysed by stirring in 60 ml guanidine lysis buffer (10 mM Tris HCl, 6 M guanidine, 100 mM NaH 2 PO 4 , pH 8.0) at room temperature for 2 h. The lysate was clarified by centrifugation and supernatant was mixed with 6 ml of Ni-resin (His-Bind resin; Novagen) overnight at 4uC. Resin was transferred to a chromatography column, washed with 10 volumes of wash buffer 2 (20 mM Tris HCl, 450 mM NaCl, 0.1% Tween, pH 8.0), and bound proteins were eluted with 3.5 column volumes of elution buffer (20 mM Tris-HCl, 450 mM NaCl, 0.1% Tween, 250 mM imidazole, pH 8.0). The eluate was dialyzed in Spectrapor 3 membrane against 2 L of dialysis buffer (4 M guanidine, 20 mM Tris, pH 8.0), overnight at 4uC.