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Titolo:
Expression of delta, kappa and mu human opioid receptors in Escherichia coli and reconstitution of the high-affinity state for agonist with heterotrimeric G proteins
Autore:
Stanasila, L; Massotte, D; Kieffer, BL; Pattus, F;
Indirizzi:
ESBS,staden,50, CNRS, Dept Recepteurs & Prot Membranaires, Illkirch Graffen ESBS Illkirch Graffenstaden France Prot Membranaires, Illkirch Graffen
Titolo Testata:
EUROPEAN JOURNAL OF BIOCHEMISTRY
fascicolo: 2, volume: 260, anno: 1999,
pagine: 430 - 438
SICI:
0014-2956(199903)260:2<430:EODKAM>2.0.ZU;2-K
Fonte:
ISI
Lingua:
ENG
Soggetto:
INFECTED INSECT CELLS; OPIATE-RECEPTOR; FUNCTIONAL EXPRESSION; BRAIN MEMBRANES; PHARMACOLOGICAL CHARACTERIZATION; CHROMOSOMAL ASSIGNMENT; CDNA CLONING; RAT-BRAIN; BINDING; SELECTIVITY;
Keywords:
opioid receptor; GTP-binding protein; expression in Escherichia coli; reconstitution; pharmacology;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
41
Recensione:
Indirizzi per estratti:
Indirizzo: Pattus, F ESBS,staden,50, CNRS, Dept Recepteurs & Prot Membranaires, Illkirch Graffen ESBS Illkirch Graffenstaden France ranaires, Illkirch Graffen
Citazione:
L. Stanasila et al., "Expression of delta, kappa and mu human opioid receptors in Escherichia coli and reconstitution of the high-affinity state for agonist with heterotrimeric G proteins", EUR J BIOCH, 260(2), 1999, pp. 430-438

Abstract

Human opioid receptors of the delta, mu and kappa subtypes were successfully expressed in Escherichia coli as fusions to the C-terminus of the periplasmic maltose-binding protein, MBP. Expression levels of correctly folded receptor molecules were comparable for the three subtypes and reached an average of 30 receptors.cell(-1) or 0.5 pmol.mg(-1) membrane protein. Binding of [H-3]diprenorphine to intact cells or membrane preparations was saturatable, with a dissociation constant, K-D, of 2.5 nM, 0.66 nM and 0.75 nM for human delta, mu and kappa opioid receptors (hDOR, hMOR and hKOR, respectively). Recombinant receptors of the three subtypes retained selectivity and nanomolar affinity for their specific antagonists. Agonist affinities were decreased by one to three orders of magnitude as compared to values measuredfor receptors expressed in mammalian cells. The effect of sodium on agonist binding to E. coli-expressed receptors was investigated. Receptor high-affinity state for agonists was reconstituted in the presence of heterotrimeric G proteins. We also report affinity values of-endomorphins 1 and 2 for mu opioid receptors expressed both in E. coli and in COS cells. Our results confirm that opioid receptors can be expressed in a functional form in bacteria and point out the advantages of E. coli as an expression system for pharmacological studies.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 28/03/20 alle ore 14:04:55