Advanced

Overexpression and functional characterisation of the human melanocortin 4 receptor in Sf9 cells

Dolby, Viveka LU ; Collén, Anna LU ; Lundqvist, A and Cronet, P (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:
author
organization
publishing date
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
2007-07-04 16:14:53
date last changed
2017-01-01 04:36:40
@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},
  keyword      = {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},
  volume       = {37},
  year         = {2004},
}