兰尼定受体
重组DNA
HEK 293细胞
标志标签
亲和层析
Myc标签
化学
链霉亲和素
蛋白质纯化
突变体
生物化学
生物素化
分子生物学
色谱法
受体
生物
融合蛋白
生物素
基因
酶
作者
Yifan Hu,Kavita A. Iyer,Ashok Kumar Nayak,Nagomi Kurebayashi,Takashi Murayama,Montserrat Samsó
出处
期刊:Bio-protocol
[Bio-Protocol]
日期:2021-08-05
卷期号:11 (15)
被引量:4
标识
DOI:10.21769/bioprotoc.4112
摘要
High quantities of purified ryanodine receptor (RyR), a large (2.26 MDa) intracellular homotetrameric membrane protein, can be obtained from heterologous expression in HEK293 cells and used for structure determination by cryo-EM. The advantage of using recombinant protein is that the variability due to post-translational modifications can be minimized, to which the high resolution of up to 2.4 Å achieved for RyR2 can be attributed ( Iyer et al., 2020 ). In addition, recombinant protein expression enables the study of mutations that are deleterious when expressed homozygously in animals. Protein purification was achieved using two strategies, sucrose density gradient and affinity chromatography, which have previously been used for purification of RyR from tissue. The sucrose gradient method was developed from ( Lee et al., 1994 ) and later adapted for cryo-EM ( Samsó et al., 2005 ). The affinity chromatography method takes advantage of the high affinity of RyR for its ligand FKBP12/12.6, by using a construct between FKBP and streptavidin binding protein (SBP) ( Cabra et al., 2016 ). While the sucrose gradient method can yield a higher protein concentration (≥ 2 mg/ml), the affinity purification method is faster. Both methods are suitable and applicable to the purification of recombinant proteins and were successfully used in the first 3D near-atomic reconstructions of RyRs purified from cells expressing disease mutants ( Iyer et al., 2020 ). This purification protocol is also suitable for functional studies, such as single-channel analysis, that require pure RyR protein.
科研通智能强力驱动
Strongly Powered by AbleSci AI