Heterologous expression and purification of the serotonin type 4 receptor from transgenic mouse retina.
Heterologous expression and purification of the serotonin type 4 receptor from transgenic mouse retina.
复制标题
转基因小鼠视网膜中 4 型血清素受体的异源表达和纯化。
DOI：
10.1021/bi8018527
复制
发表时间：
2008
期刊：
影响因子：
2.9
通讯作者：
Salon,JohnA
中科院分区：
文献类型：
--
作者：
Salom,David;Wu,Nan;Sun,Wenyu;Dong,Zhiqian;Palczewski,Krzysztof;Jordan,Steven;Salon,JohnA
最近的突破，在解决X-射线结构的G蛋白偶联受体（GPCR）与扩散配体已广泛突变或重组受体融合蛋白异源表达在常规的体外细胞为基础的系统。虽然这些进展现在表明可以实现该超家族的非视紫红质成员的结晶，但使用经根本修饰的蛋白质可能会限制衍生结构与精确指导药物设计的相关性。为了更好地使天然GPCR结构的研究，我们在这里报告的努力，工程师在体内的表达系统，利用视网膜的感光系统表达异源GPCR与天然人类序列在一个生物化学上均匀的和非功能性构象。作为一个例子，我们表明，人5 HT 4受体，当放置在小鼠视蛋白启动子和视蛋白杆外段（ROS）靶向序列的影响下，定位于转基因小鼠视网膜的ROS。免疫印迹和放射性配体结合试验表明，所得受体蛋白是均匀糖基化的，并且是完整的。在溶解后，视网膜5 HT 4受体保留了其在视网膜膜中的初始状态的结合特性。与工程化的T7单克隆表位序列，溶解的受体很容易纯化一步免疫亲和层析和纯化的受体在洗涤剂溶液中保留其配体结合特性。这种表达方法通常可用于产生功能性的高质量GPCR蛋白。
Recent breakthroughs in the solution of X-ray structures for G protein-coupled receptors (GPCRs) with diffusible ligands have employed extensively mutated or recombined receptor fusion proteins heterologously expressed in conventional in vitro cell-based systems. While these advances now show that crystallization of non-rhodopsin members of this superfamily can be accomplished, the use of radically modified proteins may limit the relevance of the derived structures for precision-guided drug design. To better enable the study of native GPCR structures, we report here efforts to engineer an in vivo expression system that harnesses the photoreceptor system of the retina to express heterologous GPCRs with native human sequences in a biochemically homogeneous and pharmacologically functional conformation. As an example, we show that the human 5HT4 receptor, when placed under the influence of the mouse opsin promoter and an opsin rod outer segment (ROS) targeting sequence, localized to ROS of transgenic mouse retina. The resulting receptor protein was uniformly glycosylated and pharmacologically intact as demonstrated by immunoblotting and radioligand binding assays. Upon solubilization, the retinal 5HT4 receptor retained the binding properties of its initial state in retinal membranes. With the engineered T7 monoclonal epitope sequence, the solubilized receptor was easily purified by one-step immunoaffinity chromatography and the purified receptor in detergent solution preserved its ligand binding properties. This expression method may prove generally useful for generating functional, high-quality GPCR protein.