   3.4.22.31 | purified native enzyme free and in complex with inhibitor E-64, hanging drop vapor diffusion technique, mixing of 10 mg/ml protein in 20 mM Tris, pH 8.0, and 20 mM NaCl with reservoir solution containing 72% w/v MPD, and 0.1 M Tris, pH 8.8, 20°C, 2 weeks, the enzyme-inhibitor complex is built from activated ananain mixed with inhibition buffer containing 20 mM sodium acetate, pH 5.5, 300 mM KCl, 1 mM EDTA, 10 mM L-cysteine, 5% v/v DMSO, with 0.5 mM E-64 at a final concentration of 0.1 mM and incubation at room temperature for 3 h. The inhibited ananain is desalted and equilibrated with 20 mM Tris, pH 8.0, and 20 mM NaCl and then concentrated to 6.5 mg/ml, crystals of E-64-ananain are obtained in the same condition as for unbound ananain, X-ray diffraction structure determination and analysis at 1.73 and 1.98 A resolution, respectively |
