活酵母生物传感器通过表位选择性蛋白酶检测肽变体。

Q2 Agricultural and Biological Sciences
生物设计研究(英文) Pub Date : 2023-03-15 eCollection Date: 2023-01-01 DOI:10.34133/bdr.0003
Tea Crnković, Benjamin J Bokor, Mead E Lockwood, Virginia W Cornish
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引用次数: 0

摘要

我们之前证明,我们可以劫持真菌信息素信号通路,以提供一种活酵母生物传感器,在该生物传感器中,肽生物标志物被G蛋白偶联受体识别,并被改造为转录读数。在这里,我们证明了蛋白酶可以重新引入生物传感器,以提供一种简单的机制来区分肽配体中的单个氨基酸变化,否则,使用基于结合的分析可能很难检测到这些变化。我们表征了五种真菌信息素G蛋白偶联受体、肽和蛋白酶的剂量-反应曲线——酿酒酵母、白色念珠菌、绒球裂殖酵母、八孢裂殖酵母和日本裂殖酵母。对这些S中最具选择性的进行丙氨酸扫描。酿酒酵母和白色念珠菌。发现了两种肽变体,其显示出蛋白酶的切割减少(CaPep2A和CaPep2A13A)。然后,通过利用具有和不具有蛋白酶的生物传感器菌株来区分这些肽,蛋白酶选择性地切割并将2个肽CaPep和CaPep13A的半最大激活所需的肽的表观浓度分别改变超过一个数量级。这些结果支持这样一种假设,即具有序列特异性蛋白酶的活酵母生物传感器可以将单个氨基酸的变化转化为半最大激活所需的肽浓度的一个以上数量级的明显变化。通过计算建模和定向进化进行进一步的工程设计,生物传感器可能会区分这里进行的丙氨酸扫描之外的各种肽序列。未来,我们设想将蛋白酶结合到我们的活酵母生物传感器中,用作护理点诊断、可扩展的通信语言和其他应用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Peptide Variant Detection by a Living Yeast Biosensor via an Epitope-Selective Protease.

Peptide Variant Detection by a Living Yeast Biosensor via an Epitope-Selective Protease.

Peptide Variant Detection by a Living Yeast Biosensor via an Epitope-Selective Protease.

Peptide Variant Detection by a Living Yeast Biosensor via an Epitope-Selective Protease.

We previously demonstrated that we could hijack the fungal pheromone signaling pathway to provide a living yeast biosensor where peptide biomarkers were recognized by G-protein-coupled receptors and engineered to transcribe a readout. Here, we demonstrated that the protease could be reintroduced to the biosensor to provide a simple mechanism for distinguishing single-amino-acid changes in peptide ligands that, otherwise, would likely be difficult to detect using binding-based assays. We characterized the dose-response curves for five fungal pheromone G-protein-coupled receptors, peptides, and proteases-Saccharomyces cerevisiae, Candida albicans, Schizosaccharomyces pombe, Schizosaccharomyces octosporus, and Schizosaccharomyces japonicus. Alanine scanning was carried out for the most selective of these-S. cerevisiae and C. albicans-with and without the protease. Two peptide variants were discovered, which showed diminished cleavage by the protease (CaPep2A and CaPep2A13A). Those peptides were then distinguished by utilizing the biosensor strains with and without the protease, which selectively cleaved and altered the apparent concentration of peptide required for half-maximal activation for 2 peptides-CaPep and CaPep13A, respectively-by more than one order of magnitude. These results support the hypothesis that the living yeast biosensor with a sequence-specific protease can translate single-amino-acid changes into more than one order of magnitude apparent shift in the concentration of peptide required for half-maximal activation. With further engineering by computational modeling and directed evolution, the biosensor could likely distinguish a wide variety of peptide sequences beyond the alanine scanning carried out here. In the future, we envision incorporating proteases into our living yeast biosensor for use as a point of care diagnostic, a scalable communication language, and other applications.

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来源期刊
CiteScore
3.90
自引率
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审稿时长
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