| Δ3-Δ2-Enoyl-CoA isomerase from the yeast Saccharomyces cerevisiae: Molecular and structural characterization | ||
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The bacterial cells were lysed by thawing the frozen cell suspension. Homogenation of the lysate was enhanced by adding DNase, RNase and lysozyme to the suspension and by incubating the mixture at +35°C for 15-30 minutes. The soluble protein-containing supernatant was separated by centrifugation. For the characterization of Eci1p (I), the recombinant protein was purified using three subsequent chromatographic steps. The columns used were DEAE Sephacel anion exchange, Resource S cation exchange and Superdex 200 HR 10/30 size exclusion columns. For the crystallization of Eci1p (II), the purification protocol was modified in order to obtain larger amounts of pure protein. Butyl sepharose was used as the first column, followed by hydroxyapatite and Poros SP columns. The purity of the protein was analyzed by sodium dodecyl sulphate polyacrylamine gel electrophoresis (SDS-PAGE) (Laemmli 1970) using 12% polyacrylamide gels and Coomassie Blue staining (Ausubel et al. 1989). The purified protein was stored at +4°C in 20 mM potassium phosphate, pH 7.2, 450 mM KCl, 1 mM EDTA, 1 mM EGTA, 0.5 mM benzamidine-HCl.