Structural aspects of nucleotide ligand binding by a bacterial 2H phosphoesterase

Myllykoski, Matti; Kursula, Petri

The cell suspension was supplemented with 0.1 mg/ml lysozyme and sonicated. The lysate was clarified by a 30-min centrifugation at 27000 g. The clarified lysate was then mixed with Ni-NTA (Qiagen) matrix. The matrix was washed with lysis buffer, and the protein was eluted from the matrix with elution buffer containing 500 mM imidazole. Fractions were analysed using SDS-PAGE. His-tagged TEV protease [36] was added to the eluted fractions containing LigT, in order to remove the His 6 tag, and this mixture was dialyzed overnight against lysis buffer devoid of imidazole. The dialyzed sample was passed through the Ni-NTA matrix to remove TEV protease, uncleaved LigT, and any other Ni-NTA binding contaminants. The eluted fractions were analyzed using SDS-PAGE. Fractions with cleaved LigT were concentrated and applied to a Superdex 75 16/60 (GE Healthcare) size exclusion chromatography column equilibrated with 10 mM Na-HEPES (pH 7.5), 100 mM NaCl, and 0.5 mM TCEP. Fractions were analysed using SDS-PAGE, and the fractions containing pure LigT were pooled. Approximately 25 mg of pure protein was obtained from one liter of expression culture. Pure LigT was flash-frozen in small batches with liquid nitrogen and stored at -70ËšC until use.