Overexpression and purification of the RyR1 pore-forming region.
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.
Kang
Gil Bu
GB
Department of Life Science, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea.
Song
Hye-Eun
HE
Song
Dong-Woo
DW
Kim
Mun-Kyoung
MK
Rho
Seong-Hwan
SH
Kim
Do Han
DH
Eom
Soo Hyun
SH
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
D003902:Detergents; D004123:Dimethylamines; D010446:Peptide Fragments; D019837:Ryanodine Receptor Calcium Release Channel; C014518:dodecyldimethylamine oxide