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פותח על ידי קלירמאש פתרונות בע"מ -
Localization and characterization of three different β‐adrenergic receptors expressed in Escherichia coli
Year:
1990
Source of publication :
European Journal of Biochemistry
Authors :
אשדת, יובל
;
.
Volume :
187
Co-Authors:
CHAPOT, M.‐P., Laboratoire de Biologie Moléculaire Des Récepteurs, Centre National de la Recherche Scientifique, Université Paris Vii, Institut Pasteur, France
ESHDAT, Y., Laboratoire de Biologie Moléculaire Des Récepteurs, Centre National de la Recherche Scientifique, Université Paris Vii, Institut Pasteur, France
MARULLO, S., Laboratoire de Biologie Moléculaire Des Récepteurs, Centre National de la Recherche Scientifique, Université Paris Vii, Institut Pasteur, France
GUILLET, J.‐G., Laboratoire de Biologie Moléculaire Des Récepteurs, Centre National de la Recherche Scientifique, Université Paris Vii, Institut Pasteur, France
CHARBIT, A., Unité de Programmation Moléculaire et Toxicologie Génétique, Centre National de la Recherche Scientifique Unité 271, Institut National de la Santé et de la Recherche Médicale U163, Paris, France
STROSBERG, A.D., Laboratoire de Biologie Moléculaire Des Récepteurs, Centre National de la Recherche Scientifique, Université Paris Vii, Institut Pasteur, France
DELAVIER‐KLUTCHKO, C., Laboratoire de Biologie Moléculaire Des Récepteurs, Centre National de la Recherche Scientifique, Université Paris Vii, Institut Pasteur, France
Facilitators :
From page:
137
To page:
144
(
Total pages:
8
)
Abstract:
After fusion with the N‐proximal portion of the outer membrane protein LamB, three β‐adrenergic receptors, the human β1‐ and β2‐ and turkey β1‐adrenergic receptor, were expressed in Escherichia coli with retention of their own specific pharmacological properties. Molecular characterization and localization of the three receptors in bacteria and comparison of the behaviour of each hybrid protein are reported. The bacteria were lysed and fractionated on a sucrose gradient. Saturable [125I]iodocyanopindolol binding activity was found associated mainly with the inner membrane fraction, suggesting that the receptor is correctly folded in this membrane. Binding activity was also found in the outer membrane fraction but varied according to the receptor type. Photoaffinity labeling experiments revealed that the receptors exhibit binding activity only after proteolytic removal of the LamB moiety from the fusion protein. The three hybrid proteins, detected in immunoblots by anti‐peptide antibodies, were found mainly in the outer membrane fraction. Each of them exhibited different susceptibility to intrinsic bacterial proteolytic enzymes; sites of proteolytic cleavage were localized by the use of anti‐peptide antibodies. The functional expression in E. coli of three β‐adrenergic receptors with similar structure but different amino acid sequences suggests that this expression system may be a general feature among similar receptors of the family of G‐protein‐coupled receptors. The level of expressed binding activity of a given receptor will be within the control of proteolytic degradation processes, depending on the primary sequence of the receptor. Constructions of new hybrid proteins, in combination with expression in protease mutants of E. coli, should help in controlling such processes. Copyright © 1990, Wiley Blackwell. All rights reserved
Note:
Related Files :
Animal
Base Sequence
cell membrane
gene expression
עוד תגיות
תוכן קשור
More details
DOI :
10.1111/j.1432-1033.1990.tb15287.x
Article number:
Affiliations:
Database:
סקופוס
Publication Type:
מאמר
;
.
Language:
אנגלית
Editors' remarks:
ID:
23262
Last updated date:
02/03/2022 17:27
Creation date:
16/04/2018 23:58
Scientific Publication
Localization and characterization of three different β‐adrenergic receptors expressed in Escherichia coli
187
CHAPOT, M.‐P., Laboratoire de Biologie Moléculaire Des Récepteurs, Centre National de la Recherche Scientifique, Université Paris Vii, Institut Pasteur, France
ESHDAT, Y., Laboratoire de Biologie Moléculaire Des Récepteurs, Centre National de la Recherche Scientifique, Université Paris Vii, Institut Pasteur, France
MARULLO, S., Laboratoire de Biologie Moléculaire Des Récepteurs, Centre National de la Recherche Scientifique, Université Paris Vii, Institut Pasteur, France
GUILLET, J.‐G., Laboratoire de Biologie Moléculaire Des Récepteurs, Centre National de la Recherche Scientifique, Université Paris Vii, Institut Pasteur, France
CHARBIT, A., Unité de Programmation Moléculaire et Toxicologie Génétique, Centre National de la Recherche Scientifique Unité 271, Institut National de la Santé et de la Recherche Médicale U163, Paris, France
STROSBERG, A.D., Laboratoire de Biologie Moléculaire Des Récepteurs, Centre National de la Recherche Scientifique, Université Paris Vii, Institut Pasteur, France
DELAVIER‐KLUTCHKO, C., Laboratoire de Biologie Moléculaire Des Récepteurs, Centre National de la Recherche Scientifique, Université Paris Vii, Institut Pasteur, France
Localization and characterization of three different β‐adrenergic receptors expressed in Escherichia coli
After fusion with the N‐proximal portion of the outer membrane protein LamB, three β‐adrenergic receptors, the human β1‐ and β2‐ and turkey β1‐adrenergic receptor, were expressed in Escherichia coli with retention of their own specific pharmacological properties. Molecular characterization and localization of the three receptors in bacteria and comparison of the behaviour of each hybrid protein are reported. The bacteria were lysed and fractionated on a sucrose gradient. Saturable [125I]iodocyanopindolol binding activity was found associated mainly with the inner membrane fraction, suggesting that the receptor is correctly folded in this membrane. Binding activity was also found in the outer membrane fraction but varied according to the receptor type. Photoaffinity labeling experiments revealed that the receptors exhibit binding activity only after proteolytic removal of the LamB moiety from the fusion protein. The three hybrid proteins, detected in immunoblots by anti‐peptide antibodies, were found mainly in the outer membrane fraction. Each of them exhibited different susceptibility to intrinsic bacterial proteolytic enzymes; sites of proteolytic cleavage were localized by the use of anti‐peptide antibodies. The functional expression in E. coli of three β‐adrenergic receptors with similar structure but different amino acid sequences suggests that this expression system may be a general feature among similar receptors of the family of G‐protein‐coupled receptors. The level of expressed binding activity of a given receptor will be within the control of proteolytic degradation processes, depending on the primary sequence of the receptor. Constructions of new hybrid proteins, in combination with expression in protease mutants of E. coli, should help in controlling such processes. Copyright © 1990, Wiley Blackwell. All rights reserved
Scientific Publication
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