   2.4.2.38 | endogenous activity (2967-fold purification) from serotype D ags1delta strain: solubilisation from crude membranes with 1% Triton X-100 (on ice, 30 min), HiPrep 16/10 Q-Sepharose chromatography with NaCl gradient elution (pH 8), activity peaks were pooled and concentrated (spin, 10000 Da cut-off), HiPrep 26/60 Sephacryl S-300 gel filtration chromatography with isocratic gradient elution (pH 8), concanavalin A-Sepharose 4B chromatography with alpha-methyl-mannoside elution (incubation over night, 4°C), HiTrap DEAE chromatorgraphy with NaCl gradient elution (pH 8), alpha-(1,3)-mannobiose agarose resin affinity chromatography with NaCl gradient (pH 6.5), activity peaks were combined and concentrated, overexpressed hexa-His tagged enzyme: solubilisation, Talon Superflow cobalt metal affinity chromatography, elution with 250 mM imidazole, pH 7.5 |
