Functional expression of rat alpha 2B-adrenoceptor in Escherichia coli.
Rat alpha 2B-adrenoceptor was expressed in Escherichia coli using 'ATG vector' containing cDNA encoding the 'non-glycosylated rat alpha 2-adrenoceptor' (RNG alpha 2). The highest receptor binding activity (using the alpha 2-adrenoceptor ligand [3H]MK 912) was found when transfected bacteria cultures were grown at 30 degrees C for about 4 h after induction with isopropyl beta-D-thiogalactopyranoside (IPTG). Saturation experiments showed that the radioligand bound to a single saturable site with a Kd of 1.42 +/- 0.09 nM and capacity of 281 +/- 6 fmol/mg protein. Binding constants of 14 compounds for the rat alpha 2B-adrenoceptor expressed in E. coli were determined and compared to the values previously obtained for the rat alpha 2B-adrenoceptor when expressed in COS cells as well as for native neonatal rat lung alpha 2B-adrenoceptors. The results indicate that when the rat alpha 2B-adrenoceptor is expressed in E. coli it retains identical ligand binding properties to those found when the receptor is present in the eukaryotic system. Expressing alpha 2B-adrenoceptors in E. coli would, therefore, seem to constitute a valid alternative in, e.g., drug screening and structure analysis of the alpha 2B-adrenoceptors.
Xia
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Department of Pharmaceutical Pharmacology, Uppsala University, Sweden.
Chhajlani
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Wikberg
J E
JE
D000317:Adrenergic alpha-Antagonists / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D001667:Binding, Competitive / Q000187:drug effects; D002460:Cell Line; D003198:Computer Simulation; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D008024:Ligands; D008168:Lung / Q000378:metabolism; D010957:Plasmids; D011807:Quinolizines / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D011942:Receptors, Adrenergic, alpha / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics; D013696:Temperature; D014162:Transfection
D000317:Adrenergic alpha-Antagonists; D008024:Ligands; D011807:Quinolizines; D011942:Receptors, Adrenergic, alpha; C054290:L 657743; D004247:DNA