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

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Ji-Hye Yun1, Kuglae Kim2, Youngjin Jung1

  • 1Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea.

Biochemical and Biophysical Research Communications
|December 3, 2014
PubMed

Insights

Optimized protocols enhance human melanocortin-4 receptor (hMC4R) expression and purification. Co-expression with mini-agouti-related protein (mini-AgRP) significantly improves hMC4R stability and yield for structural studies.

Area of Science:

  • Biochemistry
  • Structural Biology
  • Pharmacology

Background:

  • G protein-coupled receptors (GPCRs) are crucial transmembrane proteins and targets for numerous drugs.
  • Human melanocortin-4 receptor (hMC4R) is vital for energy homeostasis; mutations link to severe obesity.

Purpose of the Study:

  • To develop optimized protocols for high-yield expression and purification of hMC4R.
  • To facilitate structural studies of hMC4R by enhancing protein stability and solubility.

Main Methods:

  • Truncation of N- and C-termini of hMC4R with T4 lysozyme (T4L) insertion.
  • Co-expression of hMC4R with human mini-agouti-related protein (mini-AgRP) in Spodoptera frugiperda (Sf9) cells.
  • Analytical size exclusion chromatography (aSEC) and thermostability assays.

Main Results:

  • T4L insertion improved hMC4R solubility and stability.
  • Co-expression with mini-AgRP yielded 3-4 times more stable hMC4R.
  • hMC4R demonstrated homogeneity and stability at a melting temperature of 58°C with mini-AgRP.

Conclusions:

  • Optimized protocols, including T4L fusion and mini-AgRP co-expression, significantly enhance hMC4R yield and stability.
  • These methods accelerate structural investigations of hMC4R, a key player in energy balance.
  • Stable and homogeneous hMC4R is achievable, paving the way for further therapeutic target research.
Keywords:
Human agouti-related protein (hAgRP)Human melanocortin-4 receptor (hMC4R)SHU9119T4 lysozyme (T4L)

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