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Detection of a Peptide Biomarker by Engineered Yeast Receptors.
ACS Synthetic Biology ( IF 4.7 ) Pub Date : 2018-02-05 , DOI: 10.1021/acssynbio.7b00410
Adebola Adeniran 1 , Sarah Stainbrook 1 , John W Bostick 1 , Keith E J Tyo 1
Affiliation  

Directed evolution of membrane receptors is challenging as the evolved receptor must not only accommodate a non-native ligand, but also maintain the ability to transduce the detection of the new ligand to any associated intracellular components. The G-protein coupled receptor (GPCR) superfamily is the largest group of membrane receptors. As members of the GPCR family detect a wide range of ligands, GPCRs are an incredibly useful starting point for directed evolution of user-defined analytical tools and diagnostics. The aim of this study was to determine if directed evolution of the yeast Ste2p GPCR, which natively detects the α-factor peptide, could yield a GPCR that detects Cystatin C, a human peptide biomarker. We demonstrate a generalizable approach for evolving Ste2p to detect peptide sequences. Because the target peptide differs significantly from α-factor, a single evolutionary step was infeasible. We turned to a substrate walking approach and evolved receptors for a series of chimeric intermediates with increasing similarity to the biomarker. We validate our previous model as a tool for designing optimal chimeric peptide steps. Finally, we demonstrate the clinical utility of yeast-based biosensors by showing specific activation by a C-terminally amidated Cystatin C peptide in commercially sourced human urine. To our knowledge, this is the first directed evolution of a peptide GPCR.

中文翻译:

工程酵母受体对肽生物标志物的检测。

膜受体的定向进化是具有挑战性的,因为进化的受体不仅必须容纳非天然配体,而且还必须保持将新配体的检测转化为任何相关的细胞内组分的能力。G蛋白偶联受体(GPCR)超家族是最大的膜受体群。随着GPCR家族成员检测到广泛的配体,GPCR对于用户定义的分析工具和诊断方法的定向进化是一个非常有用的起点。这项研究的目的是确定天然检测α因子肽的酵母Ste2p GPCR的定向进化能否产生检测人肽生物标记物Cystatin C的GPCR。我们展示了进化的Ste2p以检测肽序列的通用方法。因为靶肽与α-因子明显不同,所以单个进化步骤是不可行的。我们转向了底物行走方法,并开发了一系列与生物标记物相似性更高的嵌合中间体的受体。我们验证我们先前的模型是设计最佳嵌合肽段的工具。最后,我们通过在商业来源的人尿中显示出C端酰胺化的胱抑素C肽的特异性激活,从而证明了基于酵母的生物传感器的临床实用性。据我们所知,这是肽GPCR的第一个定向进化。我们验证我们先前的模型是设计最佳嵌合肽段的工具。最后,我们通过在商业来源的人类尿液中显示出C端酰胺化的胱抑素C肽的特异性激活,从而证明了基于酵母的生物传感器的临床实用性。据我们所知,这是肽GPCR的第一个定向进化。我们验证我们先前的模型是设计最佳嵌合肽段的工具。最后,我们通过在商业来源的人尿中显示出C端酰胺化的胱抑素C肽的特异性激活,从而证明了基于酵母的生物传感器的临床实用性。据我们所知,这是肽GPCR的第一个定向进化。
更新日期:2018-02-05
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