دورية أكاديمية
Affinity purification of a chimeric nicotinic acetylcholine receptor in the agonist and antagonist bound states.
| العنوان: | Affinity purification of a chimeric nicotinic acetylcholine receptor in the agonist and antagonist bound states. |
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| المؤلفون: | Liu S; Pfizer Inc., Pfizer Global Research and Development, Groton, CT 06340, United States. shenping.liu@pfizer.com, Babcock MS, Bode J, Chang JS, Fischer HD, Garlick RL, Gill GS, Lund ET, Margolis BJ, Mathews WR, Rogers BN, Wolfe M, Groppi V, Baldwin ET |
| المصدر: | Protein expression and purification [Protein Expr Purif] 2011 Sep; Vol. 79 (1), pp. 102-10. Date of Electronic Publication: 2011 Jun 02. |
| نوع المنشور: | Journal Article |
| اللغة: | English |
| بيانات الدورية: | Publisher: Academic Press Country of Publication: United States NLM ID: 9101496 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1096-0279 (Electronic) Linking ISSN: 10465928 NLM ISO Abbreviation: Protein Expr Purif Subsets: MEDLINE |
| أسماء مطبوعة: | Publication: Orlando, FL : Academic Press Original Publication: San Diego : Academic Press, c1990- |
| مواضيع طبية MeSH: | Receptors, Nicotinic/*genetics , Receptors, Nicotinic/*isolation & purification , Recombinant Fusion Proteins/*genetics , Recombinant Fusion Proteins/*isolation & purification, Amino Acid Sequence ; Animals ; Chromatography, Affinity ; HEK293 Cells ; Humans ; Mice ; Molecular Sequence Data ; Nicotinic Agonists/metabolism ; Nicotinic Antagonists/metabolism ; Protein Binding ; Receptors, Nicotinic/metabolism ; Recombinant Fusion Proteins/metabolism ; Ultracentrifugation ; alpha7 Nicotinic Acetylcholine Receptor |
| مستخلص: | Nicotinic acetylcholine receptors (nAChRs) form ligand-gated ion channels that mediate fast signal transmission at synapses. These receptors are members of a large family of pentameric ion channels that are of active medical interest. An expression system utilizing a chimerical construct of the N-terminal extracellular ligand binding domain of alpha7 type nAChR and the C-terminal transmembrane portion of 5HT3 type receptor resulted high level of expressions. Two ligand affinity chromatography purification methods for this receptor have been developed. One method relies on the covalent immobilization of a high affinity small molecule alpha7 nAChR agonist, (R)-5-(4-aminophenyl)-N-(quinuclidin-3-yl) furan-2-carboxamide, and the other uses mono biotinylated alpha-bungarotoxin, an antagonist, that forms a quasi-irreversible complex with alpha7 nAChR. Detergent solubilized alpha7/5HT(3) chimeric receptors were selectively retained on the affinity resins and could be eluted with free ligand or biotin. The proteins purified by both methods were characterized by gel electrophoresis, mass spectra, amino acid composition analysis, and N-terminal sequence determination. These analyses confirmed the isolation of a mature alpha7/5HT(3) receptor with the signal peptide removed. These results suggest a scalable path forward to generate multi-milligram amounts of purified complexes for additional studies including protein crystallization. (Copyright © 2011 Elsevier Inc. All rights reserved.) |
| المشرفين على المادة: | 0 (Chrna7 protein, human) 0 (Chrna7 protein, mouse) 0 (Nicotinic Agonists) 0 (Nicotinic Antagonists) 0 (Receptors, Nicotinic) 0 (Recombinant Fusion Proteins) 0 (alpha7 Nicotinic Acetylcholine Receptor) |
| تواريخ الأحداث: | Date Created: 20110614 Date Completed: 20111028 Latest Revision: 20131121 |
| رمز التحديث: | 20240628 |
| DOI: | 10.1016/j.pep.2011.05.019 |
| PMID: | 21664975 |
| قاعدة البيانات: | MEDLINE |
Full Text Finder | تدمد: | 1096-0279 |
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| DOI: | 10.1016/j.pep.2011.05.019 |