Centre de Biotecnologia Molecular, Dept. d'Enginyeria Química, Universitat Politècnica de Catalunya, Terrassa 08222, Spain.
Biotechnol Prog. 2011 May-Jun;27(3):838-45. doi: 10.1002/btpr.615. Epub 2011 May 5.
Human M(3) muscarinic acetylcholine receptor (M3R), present in both the central and the peripheral nervous system, is involved in several neurodegenerative and autoimmune diseases. Recently, M3R overexpression has been suggested to play a role in certain forms of cancer, showing promise as a new potential pharmacological target. However, the lack of structural information hampered to develop a new potent selective and potent antagonist. We describe here different strategies for overexpressing functional M3R on the perspective of future biophysical studies. To achieve this goal, four tagged M3R genes were engineered and codon optimized. Different heterologous expression systems, including mammalian cells and viral transfection, were employed to overexpress M3R. Although codon optimization resulted in only twofold to threefold increase of M3R expression, we found that epitope tagging of the synthetic M3R, especially with hemagglutinin and Flag epitope tags, could improve M3R expression levels. On the other hand, viral transfection led to a yield of 27 pmol/mg protein that is the highest level reported so far for this receptor subtype in mammalian cells. Taking together several of the strategies used can help increasing M3R expression, not only to start purification efforts but also for secondary structural analysis trial and functional analyses.
人 M(3)毒蕈碱型乙酰胆碱受体 (M3R) 存在于中枢和外周神经系统中，与多种神经退行性和自身免疫性疾病有关。最近，M3R 的过度表达被认为在某些类型的癌症中发挥作用，有望成为新的潜在药理学靶点。然而，由于缺乏结构信息，开发新的有效、选择性的拮抗剂受到阻碍。我们在这里描述了不同的策略，以便从未来的生物物理研究角度来过度表达功能性 M3R。为了实现这一目标，设计并构建了四个带有标签的 M3R 基因，并进行了密码子优化。使用了不同的异源表达系统，包括哺乳动物细胞和病毒转染，来过度表达 M3R。尽管密码子优化仅导致 M3R 表达增加了两到三倍，但我们发现合成 M3R 的表位标记，特别是与血凝素和 Flag 表位标签的标记，可提高 M3R 的表达水平。另一方面，病毒转染可产生 27 pmol/mg 蛋白的产量，这是迄今为止在哺乳动物细胞中报告的该受体亚型的最高水平。综上所述，几种策略的联合使用可以帮助提高 M3R 的表达水平，不仅可以开始进行纯化工作，还可以进行二级结构分析试验和功能分析。
