Department of Chemical and Biomolecular Engineering, Tulane University, 6823 St Charles Ave, New Orleans, LA 70118, USA.
Department of Chemical Engineering, Carnegie Mellon University, 5000 Forbes Ave, Pittsburgh, PA 15213, USA.
Int J Mol Sci. 2020 Jun 26;21(12):4547. doi: 10.3390/ijms21124547.
The adenosine A receptor (AR) is the only adenosine receptor subtype to be overexpressed in inflammatory and cancer cells and therefore is considered a novel and promising therapeutic target for inflammatory diseases and cancer. Heterologous expression of AR at levels to allow biophysical characterization is a major bottleneck in structure-guided drug discovery efforts. Here, we apply protein engineering using chimeric receptors to improve expression and activity in yeast. Previously we had reported improved expression and trafficking of the chimeric AR variant using a similar approach. In this report, we constructed chimeric A/AR comprising the N-terminus and transmembrane domains from AR (residues 1-284) and the cytoplasmic C-terminus of the AR (residues 291-412). The chimeric receptor showed approximately 2-fold improved expression with a 2-fold decreased unfolded protein response when compared to wild type AR. Moreover, by varying culture conditions such as initial cell density and induction temperature a further 1.7-fold increase in total receptor yields was obtained. We observed native-like coupling of the chimeric receptor to GGpa1 in engineered yeast strains, activating the downstream, modified MAPK pathway. This strategy of utilizing chimeric receptor variants in yeast thus provides an exciting opportunity to improve expression and activity of "difficult-to-express" receptors, expanding the opportunity for utilizing yeast in drug discovery.
腺苷 A 受体 (AR) 是唯一在炎症细胞和癌细胞中过度表达的腺苷受体亚型，因此被认为是炎症性疾病和癌症的一种新的有前途的治疗靶点。在结构导向药物发现工作中，使 AR 异源表达达到允许进行生物物理特性鉴定的水平是一个主要瓶颈。在这里，我们应用嵌合受体的蛋白质工程来提高酵母中的表达和活性。此前，我们曾使用类似的方法报道了嵌合 AR 变体表达和运输的改善。在本报告中，我们构建了一种嵌合 A/AR，它包含 AR 的 N 端和跨膜结构域（残基 1-284）和 AR 的细胞质 C 端（残基 291-412）。与野生型 AR 相比，该嵌合受体的表达水平提高了约 2 倍，未折叠蛋白反应降低了 2 倍。此外，通过改变培养条件，如初始细胞密度和诱导温度，总受体产量进一步提高了 1.7 倍。我们观察到嵌合受体与工程酵母菌株中的 GGpa1 的天然样偶联，激活下游修饰的 MAPK 途径。因此，在酵母中利用嵌合受体变体的这种策略为提高“难以表达”受体的表达和活性提供了一个令人兴奋的机会，从而扩大了酵母在药物发现中的应用机会。
