Iwata Human Receptor Crystallography project, ERATO, JST, Kyoto 606-8501, Japan.
Microb Cell Fact. 2012 Jun 13;11:78. doi: 10.1186/1475-2859-11-78.
Recent successes in the determination of G-protein coupled receptor (GPCR) structures have relied on the ability of receptor variants to overcome difficulties in expression and purification. Therefore, the quick screening of functionally expressed stable receptor variants is vital.
We developed a platform using Saccharomyces cerevisiae for the rapid construction and evaluation of functional GPCR variants for structural studies. This platform enables us to perform a screening cycle from construction to evaluation of variants within 6-7 days. We firstly confirmed the functional expression of 25 full-length class A GPCRs in this platform. Then, in order to improve the expression level and stability, we generated and evaluated the variants of the four GPCRs (hADRB2, hCHRM2, hHRH1 and hNTSR1). These stabilized receptor variants improved both functional activity and monodispersity. Finally, the expression level of the stabilized hHRH1 in Pichia pastoris was improved up to 65 pmol/mg from negligible expression of the functional full-length receptor in S. cerevisiae at first screening. The stabilized hHRH1 was able to be purified for use in crystallization trials.
We demonstrated that the S. cerevisiae system should serve as an easy-to-handle and rapid platform for the construction and evaluation of GPCR variants. This platform can be a powerful prescreening method to identify a suitable GPCR variant for crystallography.
最近在确定 G 蛋白偶联受体（GPCR）结构方面的成功，依赖于受体变体克服表达和纯化困难的能力。因此，快速筛选功能表达稳定的受体变体至关重要。
我们开发了一个使用酿酒酵母的平台，用于快速构建和评估用于结构研究的功能性 GPCR 变体。该平台使我们能够在 6-7 天内完成从构建到变体评估的筛选周期。我们首先在该平台上确认了 25 种全长 A 类 GPCR 的功能性表达。然后，为了提高表达水平和稳定性，我们生成并评估了四种 GPCR（hADRB2、hCHRM2、hHRH1 和 hNTSR1）的变体。这些稳定的受体变体提高了功能活性和单分散性。最后，与最初筛选时酿酒酵母中功能性全长受体几乎没有表达相比，稳定的 hHRH1 在毕赤酵母中的表达水平提高到了 65 pmol/mg。稳定的 hHRH1 可用于结晶试验的纯化。
我们证明了酿酒酵母系统应该作为构建和评估 GPCR 变体的易于操作和快速平台。该平台可以作为一种强大的预筛选方法，用于鉴定适合结晶学研究的 GPCR 变体。
