Directed evolution of G protein-coupled receptors in yeast for higher functional production in eukaryotic expression hosts.
Directed evolution of G protein-coupled receptors in yeast for higher functional production in eukaryotic expression hosts.
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DOI：
10.1038/srep21508
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发表时间：
2016-02-25
影响因子：
4.6
通讯作者：
Plückthun A
中科院分区：
文献类型：
--
作者：
Schütz M;Schöppe J;Sedlák E;Hillenbrand M;Nagy-Davidescu G;Ehrenmann J;Klenk C;Egloff P;Kummer L;Plückthun A
尽管最近的成功，许多G蛋白偶联受体（GPCR）仍然难以详细的分子研究，由于产量不足，即使在最复杂的真核表达系统。在这里，我们介绍了一个强大的方法，采用定向进化的GPCR在酵母中，允许快速，有效地产生受体的变体，在真核表达宿主中显示出强烈增加的功能生产水平。通过在这项研究中进化出三种不同的受体，该方法具有广泛的适用性，即使对于非常难以表达的GPCR也是如此。与野生型受体相比，进化的变体在昆虫细胞中的功能性生产增加了26倍。除了产量增加之外，所获得的变体还表现出改善的生物物理特性，而功能特性基本上不受影响。因此，所提出的方法拓宽了可用于详细调查的GPCR组合。有趣的是，GPCR在酵母中的功能性生产可以通过诱导宿主适应进一步增加。
Despite recent successes, many G protein-coupled receptors (GPCRs) remained refractory to detailed molecular studies due to insufficient production yields, even in the most sophisticated eukaryotic expression systems. Here we introduce a robust method employing directed evolution of GPCRs in yeast that allows fast and efficient generation of receptor variants which show strongly increased functional production levels in eukaryotic expression hosts. Shown by evolving three different receptors in this study, the method is widely applicable, even for GPCRs which are very difficult to express. The evolved variants showed up to a 26-fold increase of functional production in insect cells compared to the wild-type receptors. Next to the increased production, the obtained variants exhibited improved biophysical properties, while functional properties remained largely unaffected. Thus, the presented method broadens the portfolio of GPCRs accessible for detailed investigations. Interestingly, the functional production of GPCRs in yeast can be further increased by induced host adaptation.