Purification of recombinant HvRNASET2 enzyme

The supernatant was concentrated by ultrafiltration using an Amicon stirred cell (Merck Millipore) equipped with a 10-kDa membrane to a final volume of 40 ml and extensively dialyzed against 20 mM sodium phosphate and 150 mM NaCl, pH 7.5. The sample was added with NaCl up to 1 M final concentration and the protein was purified using a HiTrap chelating affinity column (5 ml) (GE Healthcare) previously loaded with 100 mM NiCl₂ and equilibrated with 20 mM sodium phosphate, 1 M NaCl, pH 7.5. The column was washed with this buffer until the absorbance value at 280 nm was that of the buffer. rHvRNASET2 was eluted with the same buffer added with 100 mM imidazole; the fractions were equilibrated with 20 mM sodium phosphate and 150 mM NaCl, pH 7.5, by gel-permeation chromatography (PD10 column, GE Healthcare). The amount of protein was determined by using the absorbance intensity at 280 nm and a molar extinction coefficient of 66 mM⁻¹ cm⁻¹ (6 (link)). The recombinant rHvRNASET2 was isolated as a single band at ≈36 kDa with >90% purity as judged by SDS-PAGE analysis: ≈3.5 mg of purified enzyme per liter of fermentation broth was obtained.