   3.4.21.88 | purified recombinant His-tagged wild-type LexA and mutant enzymes S160A, K197A, and G126D, the isolated C-terminal segment and the N-domain, wild-type LexA diffraction-quality crystals grow by vapour diffusion method in about three months from a drop consisting of 0.002 ml of 6 mg/ml protein solution and 0.002 ml of 100 mM bis-Tris, pH 6.5 or pH 8.5, 100 mM NaCl or 200 mM MgCl2, 5% glycerol, 25% PEG 3350 (form I and form II, respectively). The microbatch-under-oil method is used for crystallization of the C-domain by mixing of 0.002 ml of 10 mg/ml protein solution with 0.002 ml 100 mM Tris-HCl, pH 7.5, 25% glycerol, and 40% v/v pentaerythritol ethoxylate (15/4 EO/OH). Diffraction-quality crystals of the S160A mutant are obtained by microbatch-under-oil method and mixing of 0.002 ml 10 mg/ml protein and 0.002 ml 100 mM bis-Tris, pH 6.5, 200 mM MgCl2, and 25% w/v PEG 3350 (form III), and of mutant K197A by mixing of 0.002 ml 10 mg/ml protein solution with 0.002 ml crystallization solution containing 100 mM HEPES, pH 7.5, 20 mM MgCl2, and 22% w/v polyacrylic acid sodium salt (form IV), while crystals of the G126D mutant are grown from a solution consisting of 0.002 ml of 10 mg/ml protein with 0.002 ml of 100 mM Tris-HCl, pH 8.5, 100 mM sodium acetate trihydrate, and 30% w/v PEG 4000 (form V), mutant crystals grow about 2 months, X-ray diffraction structure determination and analysis at 1.48-2.25 A resolution |
