One-Step Affinity Purification of the G Protein βγ Subunits from Bovine Brain Using a Histidine-Tagged G Protein α Subunit

An efficient one-step affinity purification of bovine brain G protein βγ subunits (βγ's) is described. The βγ's, in a detergent extract of brain membranes, are first dissociated from the α subunits (α's), reassociated with decahistidine-tagged αilproduced in bacteria, and then adsorbed onto Ni2+–nitrilotriacetic acid–agarose via the histidine tag. This mild adsorption retained the high activity of the ligand α's, in contrast to the commonly used chemical crosslinking methods. A wash step with a buffer containing chaotropic ions (SCN−) completely removed contaminating proteins that were refractory to washes with high concentrations of detergents, after which the highly purified βγ's were eluted with a buffer containing Al3+, Mg2+, and F−ions. The obtained βγ's were found to be fully functional, as assessed by their ability to support pertussis toxin-catalyzed ADP-ribosylation of αil. Since the combination of the mild adsorption via the histidine tag and the wash with chaotropic ions can be easily applied to purifying βγ's from various animal tissues, this new chromatographic method is expected to facilitate the purification of other membrane proteins that bind to Gα and/or Gαβγ.