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. 2016 Apr;147(4):353–368. doi: 10.1085/jgp.201511510

Figure 11.

Figure 11.

Effect of E. coli membrane lipids in the monodispersity of purified SteT I134V/A377T, L210Q/M229V, and WT. (A) SteT I134V/A377T was solubilized from the membrane with 1% (wt/vol) DDM and immobilized in Ni-NTA beads for affinity purification. Beads were divided and poured into four columns and washed with 5 column volumes of 0.02, 0.03, 0.04, and 0.05% (wt/vol) DDM, respectively, in each column. Each protein sample was eluted from the affinity column and subjected to gel filtration chromatography in a Superdex 200 10/30 column equilibrated with the same concentration of DDM used in the affinity purification. By gradually increasing the concentration of DDM during purification, SteT I134V/A377T became less monodisperse as a result of the delipidation effect of DDM. (B) Solubilized SteT I134V/A377T in 1% (wt/vol) DDM was immobilized in Ni-NTA beads for affinity purification. Beads were divided into three columns and washed with buffer containing no lipid or 0.05 and 0.2 mg/ml E. coli lipids, respectively, in each column, always in the presence of 0.05% (wt/vol) DDM (delipidating conditions). Each protein sample was eluted from the affinity column and subjected to gel filtration chromatography in a Superdex 200 10/30 column equilibrated with 0.05% (wt/vol) DDM and the same amount of E. coli lipids used in the affinity purification. As seen in the figure, even in the presence of delipidating conditions (washing the Ni-NTA beads with 0.05% [wt/vol] DDM), the presence of E. coli lipids kept SteT I134V/A377T monodisperse in a concentration-dependent manner. (C and D) Solubilized SteT WT (C) and L210Q/M229V (D) in 1% (wt/vol) DDM were immobilized in Ni-NTA beads for affinity purification. As in B, beads were divided into three columns and washed with buffer containing no lipid or 0.05 and 0.2 mg/ml E. coli lipids, respectively, in the presence of 0.02% and 0.05% (wt/vol) DDM for WT and L210Q/M229V, respectively. Each protein sample was eluted from the affinity column and subjected to gel filtration chromatography in a Superdex 200 10/30 column equilibrated with 0.05% (wt/vol) DDM (0.02% in the case of WT) and the same amount of E. coli lipids used in the affinity purification. Even in the presence of 0.2 mg/ml E. coli lipids (and lower concentration of DDM in the case of WT: 0.02% [wt/vol] instead of 0.05%), both SteT variants eluted in a polydisperse manner.