Membrane proteins play a valuable role in biotechnology, yet the difficulty of producing high yields of functional membrane protein limits their use in synthetic biology. The practical application of G protein-coupled receptors in whole cell biosensors, for example, is restricted to those that are functionally produced at the cell surface in the chosen host, limiting the range of detectable molecules. Here, we present a facile approach to significantly improve the yield and homogeneity of functional membrane proteins in Saccharomyces cerevisiae by altering only the choice of expression vector. Expression of a model GPCR, the human adenosine A₂a receptor, from commonly used centromeric and episomal vectors leads to low yields and cellular heterogeneity due to plasmid loss in 20–90% of the cell population. In contrast, homogeneous production of GPCR is attained using a multisite integrating vector or a novel, modified high copy vector that does not require genomic integration or addition of any selection agents. Finally, we introduce a FACS-based screen, which enables rapid isolation of cells with 4- to 15-fold increases in gene dosage and up to a 9-fold increase in functional protein yield without loss of homogeneity compared to a strain isolated through conventional, low-throughput methods. These results can be extended to improve the cellular homogeneity and yield of other membrane proteins, expanding the repertoire of useful receptors for synthetic biology applications.

中文翻译：

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调节载体的稳定性和整合频率可提高酿酒酵母中功能性GPCR的产生和均一性

膜蛋白在生物技术中起着重要作用，但是难以产生高产量的功能性膜蛋白限制了它们在合成生物学中的应用。例如，G蛋白偶联受体在全细胞生物传感器中的实际应用仅限于在选定宿主的细胞表面上功能性产生的受体，从而限制了可检测分子的范围。在这里，我们提出了一种简便的方法，只需改变表达载体的选择，即可显着提高酿酒酵母中功能膜蛋白的产量和同质性。GPCR模型的表达，人腺苷A ₂普遍使用的着丝粒和游离型载体中的一种受体导致质粒产量下降，并且导致20％至90％的细胞丢失，从而导致细胞异质性降低。相反，使用多位点整合载体或不需要基因组整合或添加任何选择剂的新型，改良的高拷贝载体可实现GPCR的均质生产。最后，我们介绍了一种基于FACS的筛选方法，与通过常规方法分离的菌株相比，该方法可快速分离基因剂量增加4至15倍，功能蛋白产量增加9倍的细胞，而不会损失同质性，低通量方法。这些结果可以扩展以改善其他膜蛋白的细胞均质性和产量，扩大合成生物学应用中有用受体的库。