Purification of the bovine nicotinic acetylcholine receptor alpha-subunit expressed in baculovirus-infected insect cells.
The bovine skeletal muscle nicotinic acetylcholine receptor alpha-subunit was produced in insect, Spodoptera frugiperda (Sf9), cells by infection with a recombinant baculovirus. The expressed alpha-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 alpha-subunit protein with Zwittergent 3-14 from the Triton X-100-insoluble fraction, the alpha-subunit protein was purified by concanavalin A-Sepharose chromatography and DEAE ion-exchange chromatography. A milligram quantity of the alpha-subunit protein could be purified from 8 g (wet weight) of Sf9 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 alpha-subunit protein is homogeneous. The purified alpha-subunit protein had a high affinity for 125I-alpha-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.
Iizuka
M
M
Department of Medical Chemistry, Kyoto University Faculty of Medicine.
Fukuda
K
K
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002038:Bungarotoxins / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006031:Glycosylation; D009036:Moths; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification; D012995:Solubility
D002038:Bungarotoxins; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins