One-step affinity purification of the G protein betagamma subunits from bovine brain using a histidine-tagged G protein alpha subunit.
An efficient one-step affinity purification of bovine brain G protein betagamma subunits (betagamma's) is described. The betagamma's, in a detergent extract of brain membranes, are first dissociated from the alpha subunits (alpha's), reassociated with decahistidine-tagged alphail produced in bacteria, and then adsorbed onto Ni2+-nitrilotriacetic acid-agarose via the histidine tag. This mild adsorption retained the high activity of the ligand alpha'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 betagamma's were eluted with a buffer containing Al3+, Mg2+, and F- ions. The obtained betagamma's were found to be fully functional, as assessed by their ability to support pertussis toxin-catalyzed ADP-ribosylation of alphail. Since the combination of the mild adsorption via the histidine tag and the wash with chaotropic ions can be easily applied to purifying betagamma's from various animal tissues, this new chromatographic method is expected to facilitate the purification of other membrane proteins that bind to Galpha and/or Galphabetagamma.
Mitsubishi Kasei Institute of Life Sciences, Tokyo, Machida, 194-8511, Japan.
T
T
Tanaka
M
M
Kubota
K
K
Samizo
Y
Y
Nakajima
M
M
Hoshino
T
T
Kohno
K
K
Wakamatsu
D000327:Adsorption; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification*; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000302:isolation & purification*
D007527:Isoenzymes; D009419:Nerve Tissue Proteins; D019204:GTP-Binding Proteins