کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2020849 1069213 2011 9 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Affinity purification of a chimeric nicotinic acetylcholine receptor in the agonist and antagonist bound states
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Affinity purification of a chimeric nicotinic acetylcholine receptor in the agonist and antagonist bound states
چکیده انگلیسی

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 alpha7/5HT3 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 alpha7/5HT3 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.


► A system established resulted in high expression level of an alpha7 type chimerical receptor.
► The receptor was purified in either an agonist bound form or an antagonist bound form.
► Purified receptor has been confirmed to be the intact mature receptor by mutiple methods.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 79, Issue 1, September 2011, Pages 102–110
نویسندگان
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