1.13.12.9 | cell disruption by sonication, centrifugation, supernatant applied to HisTrap HP column equilibrated with 20 mM Tris-HCl (pH 8.0) containing 500 mM NaCl, elution with linear gradient of imidazole from 0-0.5 M, ammonium sulfate added to eluted fractions, centrifugation of precipitate, solution in 20 mM Tris-HCl (pH 8.0) containing 300 mM ammonium sulfate, several chromatographic steps using HiTrap Phenyl FF, Resource Q, and HiLoad 16/60 Superdex 200 prep grade, activated enzyme purified by HiTrap Q HP and gel filtration |