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.
authors.affiliation: Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea.
authors.forename: Ji-Hye
authors.initials: JH
authors.lastname: Yun
authors.affiliation: Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea.
authors.forename: Kuglae
authors.initials: K
authors.lastname: Kim
authors.affiliation: Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea.
authors.forename: Youngjin
authors.initials: Y
authors.lastname: Jung
authors.affiliation: Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea.
authors.forename: Jae-Hyun
authors.initials: JH
authors.lastname: Park
authors.affiliation: Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea.
authors.forename: Hyun-Soo
authors.initials: HS
authors.lastname: Cho
authors.affiliation: Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea. Electronic address: wlee@spin.yonsei.ac.kr.
authors.forename: Weontae
authors.initials: W
authors.lastname: Lee
mesh_terms: D054369:Agouti-Related Protein / Q000096:biosynthesis*; D000595:Amino Acid Sequence; D000818:Animals; D017122:Bacteriophage T4 / Q000201:enzymology; D002460:Cell Line; D002845:Chromatography; D002850:Chromatography, Gel; D005786:Gene Expression Regulation; D006801:Humans; D007313:Insecta; D008024:Ligands; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry; D009154:Mutation; D017434:Protein Structure, Tertiary; D044105:Receptor, Melanocortin, Type 4 / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis; D015398:Signal Transduction; D012995:Solubility; D013696:Temperature
chemical_list: C515198:AGRP protein, human; D054369:Agouti-Related Protein; D008024:Ligands; C470072:MC4R protein, human; D044105:Receptor, Melanocortin, Type 4; D011994:Recombinant Proteins; D009113:Muramidase
keywords: Human agouti-related protein (hAgRP); Human melanocortin-4 receptor (hMC4R); SHU9119; T4 lysozyme (T4L)