G protein-coupled receptors (GPCRs) represent the largest family of transmembrane signaling proteins, and they are considered major targets of approximately half of all therapeutic agents. Human melanocortin-4 receptor (hMC4R) plays an important role in the control of energy homeostasis, and its mutants are directly related to severe human obesity. Here, we describe optimized protocols for the high-yield expression and purification of hMC4R that will accelerate structural study. Truncations of the N- and C-termini of hMC4R with T4 lysozyme (T4L) insertion increase the solubility as well as stability of the protein. Strikingly, co-expression of human mini-agouti-related protein (mini-AgRP) in Spodoptera frugiperda (Sf9) cells enables excellent stability of hMC4R. The protein yield in the human mini-AgRP co-expression system is increased by about 3-4 times compared to that of hMC4R alone. Data from analytical size exclusion chromatography (aSEC) and thermostability assay show that hMC4R becomes homogeneous and stable with a melting temperature of 58 °C in the presence of human mini-AgRP.
|Number of pages||6|
|Journal||Biochemical and Biophysical Research Communications|
|Publication status||Published - 2015 Jan 2|
Bibliographical noteFunding Information:
We would like to thank Professor Kurt Wüthrich and Raymond Stevens for their fruitful discussions on this research. This work was supported by the Mid-career Researcher Program ( NRF-2013R1A2A2A01068963 ) through an NRF grant funded by the Ministry of Education and Science Technology , Korea. J. H. Yun is a recipient of the Brain Korea Plus (BK+) grant.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology