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

doi:10.1016/j.bbrc.2014.11.044

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

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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 language	English
Pages (from-to)	116-121
Number of pages	6
Journal	Biochemical and Biophysical Research Communications
Volume	456
Issue number	1
DOIs	https://doi.org/10.1016/j.bbrc.2014.11.044
Publication status	Published - 2015 Jan 2

Bibliographical note

Funding 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.

Publisher Copyright:
© 2014 Elsevier Inc. All rights reserved.

All Science Journal Classification (ASJC) codes

Biophysics
Biochemistry
Molecular Biology
Cell Biology

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This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/j.bbrc.2014.11.044

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title = "Co-expression of human agouti-related protein enhances expression and stability of human melanocortin-4 receptor",

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.",

author = "Yun, {Ji Hye} and Kuglae Kim and Youngjin Jung and Park, {Jae Hyun} and Cho, {Hyun Soo} and Weontae Lee",

note = "Funding Information: We would like to thank Professor Kurt W{\"u}thrich 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. Publisher Copyright: {\textcopyright} 2014 Elsevier Inc. All rights reserved.",

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AU - Cho, Hyun Soo

AU - Lee, Weontae

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N2 - 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.

AB - 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.

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

Abstract

Bibliographical note

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