Preparation of stable isotope-labeled peripheral cannabinoid receptor CB2 by bacterial fermentation
Preparation of stable isotope-labeled peripheral cannabinoid receptor CB2 by bacterial fermentation
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DOI：
10.1016/j.pep.2009.12.011
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发表时间：
2010-04-01
影响因子：
1.6
通讯作者：
Yeliseev, Alexei
中科院分区：
文献类型：
--
作者：
Berger, Christian;Ho, Jenny T. C.;Yeliseev, Alexei
我们开发了一种细菌发酵方案，用于生产稳定同位素标记的大麻素受体 CB2，以便通过核磁共振波谱对该蛋白质进行后续结构研究。人外周大麻素受体在大肠杆菌中表达为与麦芽糖结合蛋白和两个亲和标签的融合体。发酵在由矿物盐、葡萄糖和 N-15(2)-L-色氨酸组成的确定成分培养基中进行，以将标记的氨基酸掺入蛋白质中。优化培养基、生长和表达条件，使得发酵过程产生约2毫克纯化的、标记的CB2/L培养基。通过对纯化的 CB2 进行质谱表征，我们确定色氨酸中的两个 N-15 原子之一已掺入重组蛋白中。 N-15 化学位移的 NMR 分析强烈表明 N-15 原子位于色氨酸吲哚环中。重要的是，对纯化蛋白的 CNBr 裂解衍生的肽的分析证实，至少 95% 的标记色氨酸掺入到 CB2 序列中。标记的 CB2 以 1:500 的蛋白质与脂质摩尔比纯化并重构为脂质体，通过体外偶联测定中同源 G 蛋白的激活证实其具有功能。据我们所知，这是首次报道通过细菌发酵生产具有生物活性、稳定同位素标记的 G 蛋白偶联受体。由爱思唯尔公司出版
We developed a bacterial fermentation protocol for production of a stable isotope-labeled cannabinoid receptor CB2 for subsequent structural studies of this protein by nuclear magnetic resonance spectroscopy. The human peripheral cannabinoid receptor was expressed in Escherichia coli as a fusion with maltose binding protein and two affinity tags. The fermentation was performed in defined media comprised of mineral salts, glucose and N-15(2)-L-tryptophan to afford incorporation of the labeled amino acid into the protein. Medium, growth and expression conditions were optimized so that the fermentation process produced about 2 mg of purified, labeled CB2/L of culture medium. By performing a mass spectroscopic characterization of the purified CB2, we determined that one of the two N-15 atoms in tryptophan was incorporated into the recombinant protein. NMR analysis of N-15 chemical shifts strongly suggests that the N-15 atoms are located in Trp-indole rings. Importantly, analysis of the peptides derived from the CNBr cleavage of the purified protein confirmed a minimum of 95% incorporation of the labeled tryptophan into the CB2 sequence. The labeled CB2, purified and reconstituted into liposomes at a protein-to-lipid molar ratio of 1:500, was functional as confirmed by activation of cognate G proteins in an in vitro coupled assay. To our knowledge, this is the first reported production of a biologically active, stable isotope-labeled G protein-coupled receptor by bacterial fermentation. Published by Elsevier Inc.