Co-expression of human agouti-related protein enhances expression and stability of human melanocortin-4 receptor

Ji Hye Yun, Kuglae Kim, Youngjin Jung, Jae Hyun Park, Hyun Soo Cho, Weon Tae Lee

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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 languageEnglish
Pages (from-to)116-121
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume456
Issue number1
DOIs
Publication statusPublished - 2015 Jan 2

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All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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