Figure 1. Scheme of an N-terminally double-tagged protein
GST: Glutathione-S-Transferase; HIS: poly-histidine; TEV site: cleavage site for TEV protease
The pellet was lysed using the resuspension buffer (PBS, lysozyme 1mg/ml, DNase 1g/ml, 5 mM MgCl2), incubated for 30 min under agitation and the supernatant was recovered by centrifugation (3 min at 13,000 rpm).
Double affinity purification using Vivapure columns
The Vivapure 8-to-96 well Cobalt-Chelate kit (Vivascience AG) was used for a 2-step purification of the target protein. A scheme of the purification protocol is shown in Figure 2.
Briefly, an empty Vivaclear plus column was loaded with 150 l of glutathione sepharose resin and washed three times with 500 l of PBS before addition of the cell supernatant. The flow rate of the solutions through the column was speeded up by a centrifugation step (1 min at 6,000 rpm). The flow-through was discarded, the column was washed three times with 500 l of PBS, and the GST-tagged protein bound to the glutathione sepharose was eluted with 350 l of 20 mM Tris-HCl plus 10 mM reduced glutathione. The fusion protein was incubated for 2 h at 30C in the presence of TEV protease (1:100). The digested fraction was loaded onto a Vivapure Cobalt-Chelate column equilibrated with 500 l of 20 mM Tris-HCl. The target protein was recovered in the flow-through.
The column dead volume was considered to optimize the yield, namely the first 50 l of eluate was discarded and additional 100 l of Tris-HCl was loaded on top of the protein fraction. The fusion-tag was bound by the cobalt chelates in the column.
The fractions corresponding to the ly