title: Overexpression and purification of the RyR1 pore-forming region.
abstract: Ryanodine receptor 1 (RyR1) is a large homotetrameric calcium channel that plays a pivotal role in skeletal muscle contraction. Sequence comparison and mutagenesis studies indicate that the pore architecture of RyR1, including the last two transmembrane helices and the luminal loop linking them, is similar to that of the bacterial KcsA K(+) channel. Here, we describe the overexpression and purification of the C-terminal polyhistidine-tagged RyR1 pore-forming region. The nonionic detergent lauryldimethylamine oxide (LDAO) was selected for solubilization of the protein based on its ability to extract the protein from the membrane and to maintain it in a monodisperse state. The protein was then purified using nickel-affinity chromatography and gel filtration. Gel filtration analysis confirmed that the RyR1 fragment containing the pore-forming region (amino acids 4829-5037) is sufficient to form a tetramer.
authors.affiliation: Department of Life Science, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea.
authors.forename: Gil Bu
authors.initials: GB
authors.lastname: Kang
authors.forename: Hye-Eun
authors.initials: HE
authors.lastname: Song
authors.forename: Dong-Woo
authors.initials: DW
authors.lastname: Song
authors.forename: Mun-Kyoung
authors.initials: MK
authors.lastname: Kim
authors.forename: Seong-Hwan
authors.initials: SH
authors.lastname: Rho
authors.forename: Do Han
authors.initials: DH
authors.lastname: Kim
authors.forename: Soo Hyun
authors.initials: SH
authors.lastname: Eom
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003902:Detergents; D004123:Dimethylamines; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000302:isolation & purification; D020836:Protein Structure, Quaternary; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000096:biosynthesis* / Q000302:isolation & purification; D012995:Solubility
chemical_list: D003902:Detergents; D004123:Dimethylamines; D010446:Peptide Fragments; D019837:Ryanodine Receptor Calcium Release Channel; C014518:dodecyldimethylamine oxide