Overexpression and functional characterisation of the human melanocortin 4 receptor in Sf9 cells
(2004) In Protein Expression and Purification 37(2). p.455-461- Abstract
- The human melanocortin 4 receptor (MC4r) was successfully expressed in Sf9 cells using the baculovirus infection system. N- and C-terminally His-tagged receptors generated B-max values of 14 and 23 pmol receptor/mg membrane protein, respectively. The highest expression level obtained with the C-terminally His-tagged MC4r corresponded to 0.25 mg active receptor/litre culture volume. Addition of a viral signal peptide at the N-terminus of the His-tagged MC4r did not improve the expression level. Confocal laser microscopy studies revealed that both the N- and C-terminally tagged MC4r did not accumulate intracellularly and were mainly located in the plasma membrane. The recombinant receptors showed similar affinity for the agonist NDP-MSH (K-d... (More)
- The human melanocortin 4 receptor (MC4r) was successfully expressed in Sf9 cells using the baculovirus infection system. N- and C-terminally His-tagged receptors generated B-max values of 14 and 23 pmol receptor/mg membrane protein, respectively. The highest expression level obtained with the C-terminally His-tagged MC4r corresponded to 0.25 mg active receptor/litre culture volume. Addition of a viral signal peptide at the N-terminus of the His-tagged MC4r did not improve the expression level. Confocal laser microscopy studies revealed that both the N- and C-terminally tagged MC4r did not accumulate intracellularly and were mainly located in the plasma membrane. The recombinant receptors showed similar affinity for the agonist NDP-MSH (K-d = 11 nM) as to MC4r expressed in mammalian cells. Functional coupling of the highest expressed C-terminal tagged receptor to endogenous Galpha protein was demonstrated through 6TPgammaS binding upon agonist stimulation of the receptor. K-i values for the ligands MTII, HS014, alpha-, beta-, and gamma-MSH are comparable to the values obtained for MC4r expressed in mammalian cells. (C) 2004 Elsevier Inc. All rights reserved. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/138705
- author
- Dolby, Viveka LU ; Collén, Anna LU ; Lundqvist, A and Cronet, P
- organization
- publishing date
- 2004
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Insect cells, Functional characterisation, Melanocortin receptor 4, Overproduction, GPCR, Membrane protein
- in
- Protein Expression and Purification
- volume
- 37
- issue
- 2
- pages
- 455 - 461
- publisher
- Academic Press
- external identifiers
-
- wos:000224166400025
- pmid:15358370
- scopus:4444298520
- ISSN
- 1046-5928
- DOI
- 10.1016/j.pep.2004.06.018
- language
- English
- LU publication?
- yes
- id
- 5e521d9b-bdd0-4485-9e68-beca70dcde84 (old id 138705)
- date added to LUP
- 2016-04-01 11:51:36
- date last changed
- 2022-04-28 21:06:40
@article{5e521d9b-bdd0-4485-9e68-beca70dcde84, abstract = {{The human melanocortin 4 receptor (MC4r) was successfully expressed in Sf9 cells using the baculovirus infection system. N- and C-terminally His-tagged receptors generated B-max values of 14 and 23 pmol receptor/mg membrane protein, respectively. The highest expression level obtained with the C-terminally His-tagged MC4r corresponded to 0.25 mg active receptor/litre culture volume. Addition of a viral signal peptide at the N-terminus of the His-tagged MC4r did not improve the expression level. Confocal laser microscopy studies revealed that both the N- and C-terminally tagged MC4r did not accumulate intracellularly and were mainly located in the plasma membrane. The recombinant receptors showed similar affinity for the agonist NDP-MSH (K-d = 11 nM) as to MC4r expressed in mammalian cells. Functional coupling of the highest expressed C-terminal tagged receptor to endogenous Galpha protein was demonstrated through 6TPgammaS binding upon agonist stimulation of the receptor. K-i values for the ligands MTII, HS014, alpha-, beta-, and gamma-MSH are comparable to the values obtained for MC4r expressed in mammalian cells. (C) 2004 Elsevier Inc. All rights reserved.}}, author = {{Dolby, Viveka and Collén, Anna and Lundqvist, A and Cronet, P}}, issn = {{1046-5928}}, keywords = {{Insect cells; Functional characterisation; Melanocortin receptor 4; Overproduction; GPCR; Membrane protein}}, language = {{eng}}, number = {{2}}, pages = {{455--461}}, publisher = {{Academic Press}}, series = {{Protein Expression and Purification}}, title = {{Overexpression and functional characterisation of the human melanocortin 4 receptor in Sf9 cells}}, url = {{http://dx.doi.org/10.1016/j.pep.2004.06.018}}, doi = {{10.1016/j.pep.2004.06.018}}, volume = {{37}}, year = {{2004}}, }