Recombinant Human Melatonin Receptor MT1 Isolated in Mixed Detergents Shows Pharmacology Similar to That in Mammalian Cell Membranes

The human melatonin MT1 receptor—belonging to the large family of G protein-coupled receptors (GPCRs)—plays a key role in circadian rhythm regulation and is notably involved in sleep disorders and depression. Structural and functional information at the molecular level are highly desired for fine characterization of this receptor; however, adequate techniques for isolating soluble MT1 material suitable for biochemical and biophysical studies remain lacking. Here we describe the evaluation of a panel of constructs and host systems for the production of recombinant human MT1 receptors, and the screening of different conditions for their solubilization and purification. Our findings resulted in the establishment of an original strategy using a mixture of Fos14 and CHAPS detergents to extract and purify a recombinant human MT1 from Pichia pastoris membranes. This procedure enabled the recovery of relatively pure, monomeric and ligand-binding active MT1 receptor in the near-milligram range. A comparative study based on extensive ligand-binding characterization highlighted a very close correlation between the pharmacological profiles of MT1 purified from yeast and the same receptor present in mammalian cell membranes. The high quality of the purified MT1 was further confirmed by its ability to activate its cognate Gαi protein partner when reconstituted in lipid discs, thus opening novel paths to investigate this receptor by biochemical and biophysical approaches.人类褪黑素MT1受体属于G蛋白偶联受体（G protein-coupled receptors, GPCRs）大家族，在昼夜节律调控中发挥关键作用，且与睡眠障碍、抑郁症显著相关。目前学界亟需获取该受体在分子层面的结构与功能信息以实现精准表征，但仍缺乏能够分离出适用于生化与生物物理研究的可溶性MT1样品的成熟技术。本文评估了一系列构建载体与宿主系统，用于重组表达人源MT1受体，并筛选了该受体的溶解与纯化的多种条件。研究结果确立了一套原创策略：使用Fos14与CHAPS去垢剂的混合体系，从毕赤酵母（Pichia pastoris）膜中提取并纯化重组人源MT1受体。该工艺可回收获得接近毫克级、纯度较高的单体形式且具备配体结合活性的MT1受体。基于广泛配体结合表征的比较研究表明，从酵母中纯化的MT1受体与哺乳动物细胞膜上的天然受体，其药理学特征高度相似。纯化得到的MT1受体在重构入脂质盘后，能够激活其同源Gαi蛋白伴侣，进一步证实了其高活性，这为通过生化与生物物理方法研究该受体开辟了全新路径。