Expression, purification and characterization of TMCO1 for structural studies
Expression, purification and characterization of TMCO1 for structural studies
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用于结构研究的 TMCO1 表达、纯化和表征
DOI：
10.1016/j.pep.2020.105803
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发表时间：
2021-03-01
影响因子：
1.6
通讯作者：
OuYang, Bo
中科院分区：
文献类型：
--
作者：
Zhang, Ningning;Tang, Meng;OuYang, Bo
跨膜和卷曲螺旋结构域1（TMCO 1）在物种间具有高度保守的氨基酸序列，表明TMCO 1在细胞生理学中的关键作用。人类中TMCO 1的缺乏与面胸发育不良（CFTD）、青光眼、骨生成和癌症的发生有关。TMCO 1最近被鉴定为内质网（ER）Ca 2+负荷激活的Ca 2+（CLAC）释放通道，其防止ER Ca 2+过载并维持ER中的钙稳态。然而，TMCO 1通道的分子功能的结构基础仍然是难以捉摸的。为了确定TMCO 1的结构，我们筛选了TMCO 1在大肠杆菌和昆虫细胞表达系统中的表达。在大肠杆菌中成功表达了盘基网柄藻TMCO 1（DdTMCO 1）。通过亲和层析和分子排阻层析获得了纯化的重组蛋白。DPC胶束中DdTMCO 1的溶液NMR显示出三个α-螺旋跨膜区域。
Transmembrane and coiled-coil domains 1 (TMCO1) has a highly conserved amino acid sequence among species, indicating a critical role of TMCO1 in cell physiology. The deficiency of TMCO1 in humans is associated with cerebrofaciothoracic dysplasia (CFTD), glaucoma, osteogenesis and the occurrence of cancer. TMCO1 was recently identified as an endoplasmic reticulum (ER) Ca2+ load-activated Ca2+ (CLAC) release channel, which prevents ER Ca2+ overload and maintains calcium homeostasis in the ER. However, the structural basis of the molecular function of TMCO1 channel remains elusive. To determine the structure of TMCO1, we screened the expression of TMCO1 in Escherichia coli and insect cell expression systems. TMCO1 from Dictyostelium discoideum (DdTMCO1) was successfully expressed in Escherichia coli with a high yield. The pure recombinant protein was obtained by affinity chromatography and size exclusion chromatography. The solution NMR of DdTMCO1 in DPC micelles showed three alpha-helical transmembrane regions.