Efficient cell-free production of olfactory receptors: Detergent optimization, structure, and ligand binding analyses
Efficient cell-free production of olfactory receptors: Detergent optimization, structure, and ligand binding analyses
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DOI：
10.1073/pnas.0804766105
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发表时间：
2008-10-14
影响因子：
11.1
通讯作者：
Zhang, Shuguang
中科院分区：
文献类型：
--
作者：
Kaiser, Liselotte;Graveland-Bikker, Johanna;Zhang, Shuguang
在异源细胞系统中高水平生产膜蛋白，特别是G蛋白偶联受体（GPCRs），由于其跨膜结构域的固有疏水性而遇到许多困难，这经常导致蛋白聚集和细胞毒性，从而降低蛋白产量。无细胞蛋白质合成的最新进展规避了这些问题，以有时超过基于细胞的方法的产率生产膜蛋白。在这里，我们报告使用小麦胚芽提取物无细胞生产的人嗅觉受体17-4（hOR 17 -4）。利用这种简单的方法，我们还成功地产生了两个额外的嗅觉受体。为了获得可溶性嗅觉受体并提高产量，我们直接向无细胞反应中加入不同浓度的不同去污剂。为了确定一个纯化缓冲液系统，保持受体在一个非聚集的形式，我们开发了一种方法，使用小体积的尺寸排阻柱层析结合快速和灵敏的斑点印迹检测。评价了不同缓冲液组分（包括盐浓度、各种去污剂和去污剂浓度以及还原剂及其浓度）维持无细胞产生的蛋白质稳定和非聚集的能力。纯化的嗅觉受体显示出典型的α-螺旋CD光谱。表面等离子体共振测量用于显示已知配体十一醛与hOR 17 -4的结合。我们的方法产生高产量的纯化的嗅觉受体是一个里程碑，获得大量的嗅觉受体的仿生传感器的设计。此外，这种简单的方法不仅可以广泛用于其他类型的GPCR，而且可以用于其他膜蛋白。
High-level production of membrane proteins, particularly of G protein-coupled receptors (GPCRs) in heterologous cell systems encounters a number of difficulties from their inherent hydrophobicity in their transmembrane domains, which frequently cause protein aggregation and cytotoxicity and thus reduce the protein yield. Recent advances in cell-free protein synthesis circumvent those problems to produce membrane proteins with a yield sometimes exceeding the cell-based approach. Here, we report cell-free production of a human olfactory receptor 17-4 (hOR17-4) using the wheat germ extract. Using the simple method, we also successful produced two additional olfactory receptors. To obtain soluble olfactory receptors and to increase yield, we directly added different detergents in varying concentrations to the cell-free reaction. To identify a purification buffer system that maintained the receptor in a nonaggregated form, we developed a method that uses small-volume size-exclusion column chromatography combined with rapid and sensitive dot-blot detection. Different buffer components including salt concentration, various detergents and detergent concentration, and reducing agent and its concentrations were evaluated for their ability to maintain the cell-free produced protein stable and nonaggregated. The purified olfactory receptor displays a typical a alpha-helical CD spectrum. Surface plasmon resonance measurements were used to show binding of a known ligand undecanal to hOR17-4. Our approach to produce a high yield of purified olfactory receptor is a milestone toward obtaining a large quantity of olfactory receptors for designing bionic sensors. Furthermore, this simple approach may be broadly useful not only for other classes of GPCRs but also for other membrane proteins.