Abstract
The bovine skeletal muscle nicotinic acetylcholine receptor α-subunit was produced in insect, Spodoptera frugiperda (Sf 9), cells by infection with a recombinant baculovirus. The expressed α-subunit protein could not be solubilized efficiently with Triton X-100 or sodium cholate, but could be solubilized efficiently with Zwittergent 3-14, sodium dodecyl sulfate or tris (hydroxymethyl) aminomethane dodecylsulfate. After solubilization of the α-subunit protein with Zwittergent 3-14 from the Triton X-100-insoluble fraction, the α-subunit protein was purified by concanavalin A-Sepharose chromatography and DEAE ion-exchange chromatography. A milligram quantity of the α-subunit protein could be purified from 8g (wet weight) of Sf 9 cells infected with the recombinant baculovirus. Chromatographic analyses including hydroxyapatite chromatography, DEAE ion-exchange chromatography, gel filtration chromatography and chromatofocusing and sucrose density gradient centrifugation analysis suggest that the purified α-subunit protein is homogeneous. The purified α-subunit protein had a high affinity for 125I-α-bungarotoxin and was glycosylated with a high mannose-type N-linked oligosaccharide side chain. These results indicate that purification of ion channel proteins produced by the baculovirus expression system is a promising approach to structural analysis of ion channel proteins, which are extremely rare membrane proteins in native tissues.
