*4.4. Protein Purification*

Proteins were purified with Äkta purifier 900 chromatography systems (GE Healthcare, Helsinki, Finland). Cell-free extracts were loaded onto a Ni2+ Chelating HiTrapTM HP column (1–5 mL) (GE Healthcare, Finland) equilibrated with 50 mM potassium phosphate buffer, pH 7.2, at 1.0 mL/min. The column was washed with at least 3 volumes of the same buffer. Then the bound proteins were eluted with 0.5 M imidazole in 50 mM potassium phosphate buffer, pH 7.2. The fractions containing the purified enzyme were combined and dialyzed against the 50 mM potassium phosphate buffer, pH 7.2, at 4 ◦C overnight. Proteins were analyzed by SDS-PAGE according to Laemmli [66]. Protein concentration was determined by the Lowry method [67].
