High-level expression and purification of the second transmembrane domain of wild-type and mutant human melanocortin-4 receptor for solid-state NMR structural studies.
It has been demonstrated that human melanocortin-4 receptor (hMC4R) plays an important role in the control of energy homeostasis, and heterozygous mutations in the hMC4R gene are the most frequent genetic cause of severe human obesity. In order to obtain additional insight into the structure and function, we cloned, expressed, and purified the second transmembrane domain of the wild-type hMC4R (wt-TM2) and D90N mutant hMC4R (m-TM2). To facilitate structural studies of these hMC4R by solid-state NMR, efficient methods for the production of milligram quantities of isotopically labeled protein are necessary. However, large-scale production of most transmembrane proteins has been limited by experimental adversities due to insufficient yields and low solubility of protein. Nevertheless, through the optimization of the expression and purification approach, we could obtain uniformly or selectively labeled fusion proteins in yields as high as 200-250 mg per liter M9 minimal medium. These proteins were overexpressed in inclusion bodies as a fusion protein with ketosteroid isomerase (KSI) in Escherichia coli, and the fusion protein was purified using immobilized metal affinity chromatography under denaturing conditions. wt-/m-TM2 peptides were released from the fusion by cyanogen bromide cleavage at the Met residue and separated from the carrier KSI by size exclusion chromatography. Initial structural data obtained by solution NMR measurements of wt-/m-TM2 is also presented. The successful application to the production of the second transmembrane domain of human MC4R indicates that the method can be applied to other transmembrane proteins as well and also enable its structural and functional studies using solid-state NMR spectroscopy.
Park
Tae-Joon
TJ
Department of Chemistry, Protein Research Center for Bio-Industry, Hankuk University of Foreign Studies, Yongin, Republic of Korea.
Choi
Sung-Sub
SS
Gang
Ga-Ae
GA
Kim
Yongae
Y
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D005822:Genetic Vectors; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017434:Protein Structure, Tertiary; D044105:Receptor, Melanocortin, Type 4 / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate; D013251:Steroid Isomerases / Q000378:metabolism
C470072:MC4R protein, human; D008823:Micelles; D050505:Mutant Proteins; D010455:Peptides; D044105:Receptor, Melanocortin, Type 4; D011993:Recombinant Fusion Proteins; D012967:Sodium Dodecyl Sulfate; D013251:Steroid Isomerases