G protein coupled receptors (GPCRs) function as guanine nucleotide exchange factors (GEFs) at heterotrimeric G proteins, and conduct this role embedded in a lipid bilayer. Detergents are widely used to solubilise GPCRs for structural and biophysical analysis, but are poor mimics of the lipid bilayer and may be deleterious to protein function. Amphipathic polymers have emerged as promising alternatives to detergents, which maintain a lipid environment around a membrane protein during purification. Of these polymers, the polymethacrylate (PMA) polymers have potential advantages over the most popular styrene maleic acid (SMA) polymer, but to date have not been applied to purification of membrane proteins. Here we use a class A GPCR, neurotensin receptor 1 (NTSR1), to explore detergent-free purification using PMA. By using an NTSR1-eGFP fusion protein expressed in Sf9 cells, a range of solubilisation conditions were screened, demonstrating the importance of solubilisation temperature, pH, NaCl concentration and the relative amounts of polymer and membrane sample. PMA-solubilised NTSR1 displayed compatibility with standard purification protocols and millimolar divalent cation concentrations. Moreover, the receptor in PMA discs showed stimulation of both G_q and G_i1 heterotrimers to an extent that was greater than that for the detergent-solubilised receptor. PMA therefore represents a viable alternative to SMA for membrane protein purification and has a potentially broad utility in studying GPCRs and other membrane proteins.

G蛋白偶联受体（GPCR）在异三聚体G蛋白上起鸟嘌呤核苷酸交换因子（GEF）的作用，并嵌入脂质双层中进行这种作用。洗涤剂被广泛用于增溶GPCR，以进行结构和生物物理分析，但它们对脂质双层的模拟效果不佳，可能对蛋白质功能有害。两亲性聚合物已经成为去污剂的有前途的替代品，去污剂在纯化过程中保持膜蛋白周围的脂质环境。在这些聚合物中，聚甲基丙烯酸酯（PMA）聚合物具有优于最流行的苯乙烯马来酸（SMA）聚合物的潜在优势，但迄今为止尚未应用于膜蛋白的纯化。在这里，我们使用A类GPCR，神经降压素受体1（NTSR1）探索使用PMA的无去污剂纯化。通过使用在Sf9细胞中表达的NTSR1-eGFP融合蛋白，筛选了一系列增溶条件，证明了增溶温度，pH，NaCl浓度以及聚合物和膜样品的相对量的重要性。PMA溶解的NTSR1显示与标准纯化方案和毫摩尔二价阳离子浓度的相容性。此外，PMA盘中的受体显示出对两种G的刺激_q和_Gi异源三聚体的程度要大于去污剂溶解受体的程度。因此，PMA代表了SMA替代膜蛋白的可行方法，并且在研究GPCR和其他膜蛋白方面具有广泛的应用前景。