West, Anthony P., Jr. and Bjorkman, Pamela J. and Dougherty, Dennis A. and Lester, Henry A. (1997) Expression and Circular Dichroism Studies of the Extracellular Domain of the alpha Subunit of the Nicotinic Acetylcholine Receptor. Journal of Biological Chemistry, 272 (41). pp. 25468-25473. ISSN 0021-9258. http://resolver.caltech.edu/CaltechAUTHORS:WESjbc97
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To provide material suitable for structural studies of the nicotinic acetylcholine receptor, we have expressed and purified the NH2-terminal extracellular domain of the mouse muscle alpha subunit. Several constructs were initially investigated using Xenopus oocytes as a convenient small scale expression system. A fusion protein (alpha210GPI) consisting of the 210 NH2-terminal amino acids of the alpha subunit and a glycosylphosphatidylinositol anchorage sequence conferred surface alpha-bungarotoxin binding in oocytes. Coexpression of alpha210GPI with an analogous construct made from the delta subunit showed no evidence of heterodimer formation. The alpha210GPI protein was chosen for large scale expression in transfected Chinese hamster ovary cells. The alpha210GPI protein was cleaved from these cells and purified on an immunoaffinity column. Gel and column chromatography show that the purified protein is processed as expected and exists as a monomer. The purified protein also retains the two distinct, conformation-specific binding sites expected for the correctly folded alpha subunit. Circular dichroism studies of alpha210GPI suggest that this region of the receptor includes considerable beta-sheet secondary structure, with a small proportion of alpha-helix.
|Additional Information:||©1997 by The American Society for Biochemistry and Molecular Biology, Inc. (Received for publication, April 21, 1997, and in revised form, July 23, 1997) We thank David Penny for help with the Cell Pharm expression, Shelley Diamond for assistance with flow cytometry, Steve Mayo for assistance with the CD experiments, and Jon Lindstrom for providing mAb 210. This work was supported by National Institutes of Health Grants NS-11756 and NS-34407, the California Tobacco-related Disease Research Program, and the Howard Hughes Medical Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.|
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|Deposited On:||27 Nov 2006|
|Last Modified:||26 Dec 2012 09:18|
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