Furukawa, H., Haga, T. (January 2000) Expression of functional M2 muscarinic acetylcholine receptor in Escherichia coli. J Biochem, 127 (1). pp. 151-61. ISSN 0021-924X (Print)
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Abstract
The M2 muscarinic acetylcholine receptor mutant (M2 mutant), with a lack of glycosylation sites, a deletion in the central part of the third inner loop, and the addition of a six histidine tag at the C-terminus, was fused to maltose binding protein (MBP) at its N-terminus and expressed in Escherichia coli. The expression level was 0.2 nmol receptor per 100 ml culture, as assessed as [3H]L-quinuclidinyl benzilate ([3H]QNB) binding activity, when the BL 21 strain was cultured at 37 degrees C to a late growth phase and the expression was induced by isopropyl beta-thiogalactoside at 20 degrees C. No [3H]QNB binding activity was detected when it was not fused to MBP or when expression was induced at 37 degrees C instead of 20 degrees C. The MBP-M2 mutant expressed in E. coli showed the same ligand binding activity as the M2 mutant expressed in the Sporodoptera frugiperda (Sf9)/baculovirus system, as assessed as displacement of [(3)H]QNB with carbamylcholine and atropine. The MBP-M2 mutant was solubilized, purified with Co2+-immobilized Chelating Sepharose gel and SP-Sepharose, and then reconstituted into lipid vesicles with G protein Go or Gi1 in the presence or absence of cholesterol. The reconstituted vesicles showed GTP-sensitive high affinity binding for carbamylcholine and carbamylcholine-stimulated [35S]GTP gamma S binding activity in the presence of GDP. The proportion of high affinity sites for carbamylcholine and the extent of carbamylcholine-stimulated [(35)S]GTP gamma S binding were the same as those observed for the M2 mutant expressed in Sf9 cells and were not affected by the presence or absence of cholesterol. These results indicate that the MBP-M2 mutant expressed in E. coli has the same ability to interact with and activate G proteins as the M2 mutant expressed in Sf9, and that cholesterol is not essential for the function of the M2 muscarinic receptor.
| Item Type: | Paper |
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| Additional Information: | |
| Uncontrolled Keywords: | ATP-Binding Cassette Transporters Animals Carrier Proteins genetics Cholesterol chemistry metabolism Escherichia coli genetics Escherichia coli Proteins GTP-Binding Protein alpha Subunits Gi-Go Genetic Vectors Heterotrimeric GTP-Binding Proteins chemistry metabolism Humans Lipid Bilayers chemistry metabolism Monosaccharide Transport Proteins Mutagenesis Insertional Receptor Muscarinic M2 Receptors Muscarinic biosynthesis genetics metabolism Recombinant Fusion Proteins biosynthesis isolation & purification metabolism Solubility Swine |
| Subjects: | organism description > bacteria > escherichia coli bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein receptor |
| CSHL Authors: | |
| Communities: | CSHL labs > Furukawa lab |
| Depositing User: | Leigh Johnson |
| Date: | January 2000 |
| Date Deposited: | 06 Mar 2012 19:09 |
| Last Modified: | 05 Jan 2017 22:15 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/25172 |
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