Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 116-121 |
| Number of pages | 6 |
| Journal | Biochemical and Biophysical Research Communications |
| Volume | 456 |
| Issue number | 1 |
| DOIs | |
| State | Published - 2 Jan 2015 |
| Externally published | Yes |
UN SDGs
This output contributes to the following UN Sustainable Development Goals (SDGs)
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SDG 3 Good Health and Well-being
Keywords
- Human agouti-related protein (hAgRP)
- Human melanocortin-4 receptor (hMC4R)
- SHU9119
- T4 lysozyme (T4L)
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