FIG. 3.

Purification of DhpH-His₈ from A. nicotinovorans extracts. (A) Ni²⁺-chelating Sepharose affinity chromatography purification of DhpH-His₈ from crude extracts of A. nicotinovorans transformed with pART2-dhpH and grown in LB medium without nicotine induction. Lane 1, crude extract; lane 2, purified protein fraction, eluted with 200 mM imidazole. (B) Ni²⁺-affinity purification of DhpH-His₈ from extracts of A. nicotinovorans/pAO1 transformed with pART3-dhpH and grown in citrate minimal medium in the presence of 0.05% l-nicotine. Lane 1, crude extract; lane 2, purified protein fraction, eluted with 200 mM imidazole. The transformation of A. nicotinovorans with pART plasmids was achieved by electroporation with competent cells as described previously (6), with selection on kanamycin (140 μg ml⁻¹). E. coli was selected with 20 μg ml⁻¹ kanamycin.