Biochemical characterization of the Nocardia lactamdurans ACV synthetase
Fig 2
Ni2+ affinity chromatography purification of the Nocardia lactamdurans ACVS.
Nl ACVS was isolated from E. coli HM0079 cells and harvested after overnight expression at 18 ºC. A cell-free lysate was obtained through sonication and subsequently separated into a clear supernatant (CFL) and the pellet was resuspended in 8 M Urea (CFL (i)). The clear lysate was further purified using gravity flow in combination with a His-tag affinity chromatography, using two washing steps (W1, W2) and elution with 50, 150 and 250 mM imidazole, respectively (E1, E2, E3). Marker lane shows reference proteins corresponding to 170 and 130 kDa.