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

Protein Expression and Purification
Viveka DolbyPhilippe Cronet

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 0.25mg 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 (Kd = 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 GTPgammaS binding upon agonist stimulation of the receptor. Ki values for the ligands MTII, HS014, alpha-, beta-, and gamma-MSH are comparable to the values obtained for MC4r expressed in mammalian cells.

References

Jan 1, 1988·Annual Review of Microbiology·L K Miller
Apr 25, 1996·Biochemical and Biophysical Research Communications·H B SchiöthJ E Wikberg
Sep 1, 1996·Pharmacology & Toxicology·H B SchiöthJ E Wikberg
Dec 27, 1996·European Journal of Pharmacology·H ObermeierR Schulz
Aug 1, 1997·Naunyn-Schmiedeberg's Archives of Pharmacology·D LeopoldtB Nürnberg
Jun 18, 1998·British Journal of Pharmacology·H B SchiöthJ E Wikberg
Apr 21, 2001·Cellular and Molecular Life Sciences : CMLS·Z A Abdel-Malek
Mar 14, 2002·Brain Research. Brain Research Reviews·Helgi B Schiöth, Hajime Watanobe
May 15, 2002·European Journal of Pharmacology·Douglas J MacNeilLex H T Van der Ploeg
Aug 15, 2002·European Journal of Pharmacology·Douglas J MacNeilLex H T Van der Ploeg
Feb 15, 2003·Biochimica Et Biophysica Acta·Tony WarneGebhard F X Schertler
Feb 15, 2003·Biochimica Et Biophysica Acta·Christopher G TateD Clive Williams
Mar 18, 2004·Journal of Biochemical and Biophysical Methods·Viveka DolbyPhilippe Cronet

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Citations

Jul 25, 2009·Journal of Bioenergetics and Biomembranes·Marianna MadeoVincenza Dolce
Aug 14, 2010·Pharmacology & Therapeutics·Erich H Schneider, Roland Seifert
Sep 12, 2006·Biochemical and Biophysical Research Communications·Arun Kumar ShuklaHartmut Michel
Apr 21, 2005·European Journal of Pharmacology·Sergei KopanchukJarl E S Wikberg
Jan 4, 2008·Protein Expression and Purification·Tatsuya KatoEnoch Y Park
Dec 9, 2010·The British Journal of Sociology·Diane Richardson
May 5, 2020·Experimental Biology and Medicine·Xiao-Ming HuangFeng-Yuan Sun

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