| Δ3-Δ2-Enoyl-CoA isomerase from the yeast Saccharomyces cerevisiae: Molecular and structural characterization | ||
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Prior to crystallization, the purified Eci1p was concentrated to either 1, 2.3 or 2.5 mg/ml by centrifugal filtration in the following buffer: 20 mM potassium phosphate, pH 7.2, 450 mM KCl, 1 mM EDTA, 1 mM EGTA, 0.5 mM benzamidine-HCl. N-octyl-β -D-glucoside (10mM) was used as an additive in the 2.3 mg/ml protein solution. The crystallization conditions for the 1 and 2.3 mg/ml protein solutions were determined by the sparse-matrix screen (Jancarik & Kim 1991) using Crystal Screen and Crystal Screen II kits (Hampton Research). The crystallization was performed by the sitting and hanging-drop vapour diffusion methods by mixing equal volumes (2µl) of the protein and precipitant solutions. The screens were performed at +22°C and +4°C. The pH and the precipitant concentration of the promising conditions were further optimized.
To obtain crystals complexed with an active site ligand, octanoyl-CoA was added to a final concentration of 2 mM to protein solutions containing 1 and 2.5 mg/ml of Eci1p prior to crystallization. For the 1 mg/ml protein solution, Crystal Screens (Hampton Research) were used as described above. To obtain crystals from the 2.5 mg/ml Eci1p solution, the automated screening method described by Zeelen and co-workers (1994) using a crystallization robot was utilized. The crystallization was carried out in hanging drops as described above, and the crystallization conditions producing crystals were optimized.