Purification of the nicotinic acetylcholine receptor protein by affinity chromatography using a regioselectively modified and reversibly immobilized alpha-toxin from Naja nigricollis.
A new method of affinity chromatography purification of the detergent-solubilized nicotinic acetylcholine receptor protein (nAChR) is presented, based on the reversible coupling of a chemically monomodified alpha-toxin from Naja nigricollis to a resin. The alpha-toxin was monothiolated on the epsilon-amino group of its lysine-15 by reaction with N-succinimidly-3-(2-pyridyldithio)propionate and was covalently linked in a reversible manner to a thiopropyl-activated agarose resin by thiol-disulfide exchange. We found that 50% of the immobilized toxin molecules were effective for purifying nAChR, indicating a high accessibility of resin-bound toxins to their binding sites on the receptor protein. Purified alpha-toxin/nAChR complexes were eluted with nearly 100% recovery by reduction of disulfide bridges with dithiothreitol. nAChR solutions of high purity were obtained, as shown by polyacrylamide gel electrophoresis. A comparison was made with two other procedures of affinity chromatography using: (1) alpha-bungarotoxin from Bungarus multicinctus polymodified on several amines and covalently linked to a resin in a reversible manner, and (2) a commercial agarose resin bearing irreversibly immobilized alpha-cobrotoxin from Naja naja kaouthia. We conclude that: (1) the use of a selected regioselective linking of a peptidic ligand to a chromatography resin results in an increased efficiency of protein binding, and (2) a high yield of protein recovery is obtained via reversible covalent linking.
Ringler
P
P
Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France.
Kessler
P
P
Ménez
A
A
Brisson
A
A
D000818:Animals; D002038:Bungarotoxins / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D003039:Cobra Neurotoxin Proteins / Q000737:chemistry; D003432:Cross-Linking Reagents; D011978:Receptors, Nicotinic / Q000302:isolation & purification*; D013388:Succinimides
D002038:Bungarotoxins; D003039:Cobra Neurotoxin Proteins; D003432:Cross-Linking Reagents; D011978:Receptors, Nicotinic; D013388:Succinimides; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; C025154:alpha-cobratoxin