Improved membrane isolation in the purification of beta 2-adrenoceptors from transgenic Escherichia coli.
beta 2-Adrenoceptors (beta 2-AR) have been purified from many mammalian tissues. Unfortunately, other beta-AR subtypes expressed in the same cells are usually copurified, contaminating the preparation and interfering with subsequent investigations such as receptor characterization, ligand binding studies, immunoprecipitation, or development of anti-receptor antibodies. The advent of molecular biology techniques has facilitated the expression of beta 2-AR in cells in which no other similar molecules are present; thus, receptor purification has been simplified. beta 2-AR expressed in Escherichia coli provides a convenient source of receptor without the need for specialized culture facilities required for eukaryotic cells. The greater complexity of the gram-negative cell wall structure, however, complicates the purification of membrane-bound receptor from this source. In this report, we describe a reliable method for the partial purification of membrane-bound beta 2-AR from transgenic E. coli. Spheroplast formation followed by cell disruption and a carbonate wash procedure provided beta 2-AR bound to bacterial inner membrane in high yield.
Hill
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RA
Tropical Beef Centre, Queensland Department of Primary Industries, Australia. hillr@prose.dpi.qld.gov.au
Sillence
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MN
D002254:Carbonates / Q000494:pharmacology; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000737:chemistry* / Q000187:drug effects; D002473:Cell Wall / Q000187:drug effects; D002499:Centrifugation, Density Gradient; D004926:Escherichia coli / Q000737:chemistry* / Q000187:drug effects / Q000648:ultrastructure; D009113:Muramidase / Q000494:pharmacology; D010457:Peptidoglycan / Q000187:drug effects; D011312:Pressure; D018343:Receptors, Adrenergic, beta-2 / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D013010:Sonication; D013045:Species Specificity; D013104:Spheroplasts / Q000737:chemistry* / Q000648:ultrastructure
D002254:Carbonates; D010457:Peptidoglycan; D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins; C005686:sodium carbonate; D009113:Muramidase