Hitrap deae ff anion exchange column

Protein was extracted essentially based on procedures previously reported for ALDCs.^{13 (link)} The genome of Enterobacter cloacae CICC 10011 was used as a template to clone the gene of E.c.-ALDC (bud A) and inserted into the expression vector pET-21b. E. coli BL21 (DE3) harbouring pET-21b with the E.c.-ALDC gene were grown at 37 °C and induced for 5 h. E. coli cells were then harvested, and the pellet was resuspended in 20 mM tris(hydroxymethyl)aminomethane (Tris) buffer (pH 8.0), containing 1 mM dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA) and 20% glycerol, followed by lysis by sonication on ice at the power of 400 W for 10 min. The supernatant was loaded onto a pre-equilibrated HiTrap DEAE FF anion exchange column (GE Healthcare) in 20 mM Tris buffer (pH 8.0) containing 1 mM DTT. The column was then eluted using a linear NaCl gradient from 0 to 500 mM over 20 column volumes. Fractions containing the protein were collected, concentrated and subjected to gel filtration on a Superdex 200 16/60 column (GE Healthcare) in 20 mM Tris buffer (pH 8.0) containing 1 mM DTT. The purified protein was confirmed by SDS-PAGE, and the protein concentration was determined using the Bradford protein assay method.