Borko L'ubomír, Košt'an Július, Zahradníková Alexandra, Pevala Vladimír, Gašperík Juraj, Hostinová Eva, Urbániková L'ubica, Djinović-Carugo Kristina, Bauerová-Hlinková Vladena, Sevčík Jozef
Institute of Molecular Biology SAS, Dúbravska cesta 21, 845 51 Bratislava.
Protein Pept Lett. 2013 Nov;20(11):1211-6. doi: 10.2174/0929866511320110004.
Human ryanodine receptor 2 (hRyR2) is a calcium ion channel present in the membrane of the sarcoplasmic reticulum of cardiac myocytes that mediates release of calcium ions from the sarcoplasmic reticulum stores during excitation- contraction coupling. Disease-causing mutations of hRyR2 are clustered into N-terminal (amino acids 1-600), central (amino acids 2100-2500) and C-terminal (amino acids 3900-5000) regions. These regions are believed to be involved in regulation of channel gating. The N-terminal region of hRyR2 has been implicated in regulating basal channel activity by interaction with the central hRyR2 region. This paper reports preparation, crystallization and preliminary X-ray analysis of recombinant hRyR2(1-606) N-terminal fragment. Soluble hRyR2(1-606) was expressed in Escherichia coli. Purification conditions were optimized using thermal shift assay. The quality and stability of the sample was probed by dynamic light scattering. A monomeric protein showing over 95% purity was obtained. The protein was crystallized by the hanging drop vapor-diffusion method. Diffraction data with resolution 2.39 Å were collected and processed.
人兰尼碱受体2（hRyR2）是一种存在于心肌细胞肌浆网膜中的钙离子通道，在兴奋-收缩偶联过程中介导钙离子从肌浆网储存库中释放。hRyR2的致病突变集中在N端（氨基酸1-600）、中央（氨基酸2100-2500）和C端（氨基酸3900-5000）区域。这些区域被认为参与通道门控的调节。hRyR2的N端区域已被证明通过与hRyR2中央区域相互作用来调节通道的基础活性。本文报道了重组hRyR2（1-606）N端片段的制备、结晶及初步X射线分析。可溶性hRyR2（1-606）在大肠杆菌中表达。使用热迁移分析优化纯化条件。通过动态光散射探测样品的质量和稳定性。获得了纯度超过95%的单体蛋白。通过悬滴气相扩散法使该蛋白结晶。收集并处理了分辨率为2.39 Å的衍射数据。
