当前位置: X-MOL 学术J. Biomol. NMR › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
Efficient production of a functional G protein-coupled receptor in E. coli for structural studies
Journal of Biomolecular NMR ( IF 2.4 ) Pub Date : 2021-01-27 , DOI: 10.1007/s10858-020-00354-6
Layara Akemi Abiko 1 , Marco Rogowski 1 , Antoine Gautier 1, 2 , Gebhard Schertler 2 , Stephan Grzesiek 1
Affiliation  

G protein-coupled receptors (GPCRs) are transmembrane signal transducers which regulate many key physiological process. Since their discovery, their analysis has been limited by difficulties in obtaining sufficient amounts of the receptors in high-quality, functional form from heterologous expression hosts. Albeit highly attractive because of its simplicity and the ease of isotope labeling for NMR studies, heterologous expression of functional GPCRs in E. coli has proven particularly challenging due to the absence of the more evolved protein expression and folding machinery of higher eukaryotic hosts. Here we first give an overview on the previous strategies for GPCR E. coli expression and then describe the development of an optimized robust protocol for the E. coli expression and purification of two mutants of the turkey β1-adrenergic receptor (β1AR) uniformly or selectively labeled in 15N or 2H,15N. These mutants had been previously optimized for thermal stability using insect cell expression and used successfully in crystallographic and NMR studies. The same sequences were then used for E. coli expression. Optimization of E. coli expression was achieved by a quantitative analysis of losses of receptor material at each step of the solubilization and purification procedure. Final yields are 0.2–0.3 mg receptor per liter culture. Whereas both expressed mutants are well folded and competent for orthosteric ligand binding, the less stable YY-β1AR mutant also comprises the two native tyrosines Y5.58 and Y7.53, which enable G protein binding. High-quality 1H-15N TROSY spectra were obtained for E. coli-expressed YY-β1AR in three different functional states (antagonist, agonist, and agonist + G protein-mimicking nanobody-bound), which are identical to spectra obtained of the same forms of the receptor expressed in insect cells. NdeI and AgeI restriction sites introduced into the expression plasmid allow for the easy replacement of the receptor gene by other GPCR genes of interest, and the provided quantitative workflow analysis may guide the respective adaptation of the purification protocol.



中文翻译:

在大肠杆菌中高效生产功能性 G 蛋白偶联受体以进行结构研究

G 蛋白偶联受体 (GPCR) 是跨膜信号转导器,可调节许多关键的生理过程。自从他们发现以来,他们的分析一直受到难以从异源表达宿主中获得足够数量的高质量功能形式的受体的限制。尽管因其简单且易于同位素标记用于 NMR 研究而极具吸引力,但在大肠杆菌中功能性 GPCR 的异源表达已被证明特别具有挑战性,因为缺乏高等真核宿主的更进化的蛋白质表达和折叠机制。在这里,我们首先概述了 GPCR大肠杆菌表达的先前策略,然后描述了针对大肠杆菌的优化稳健协议的开发15 N 或2 H、15 N 中均匀或选择性标记的火鸡 β 1 -肾上腺素能受体 (β 1 AR)的两种突变体的表达和纯化。这些突变体之前已使用昆虫细胞表达进行了热稳定性优化,并成功用于晶体学和核磁共振研究。然后将相同的序列用于大肠杆菌表达。大肠杆菌的优化通过在溶解和纯化过程的每个步骤中对受体材料的损失进行定量分析来实现表达。最终产量为每升培养物 0.2–0.3 mg 受体。尽管两个表达的突变体都折叠良好并且能够进行正构配体结合,但稳定性较差的 YY-β 1 AR 突变体还包含两个天然酪氨酸 Y 5.58和 Y 7.53,它们能够结合 G 蛋白。获得了大肠杆菌表达的 YY-β 1的高质量1 H- 15 N TROSY 光谱三种不同功能状态下的 AR(拮抗剂、激动剂和激动剂 + G 蛋白模拟纳米抗体结合),这与昆虫细胞中表达的相同形式受体的光谱相同。引入表达质粒的 NdeI 和 AgeI 限制性位点允许其他感兴趣的 GPCR 基因轻松替换受体基因,并且提供的定量工作流程分析可以指导纯化协议的相应调整。

更新日期:2021-01-28
down
wechat
bug