Biochemical and Biophysical Research Communications 2015-01-02

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, Weontae Lee

Index: Biochem. Biophys. Res. Commun. 456(1) , 116-21, (2014)

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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.Copyright © 2014 Elsevier Inc. All rights reserved.