PDF(Pubmed)
Abstract：
Protein-protein interactions play an important biological role in every aspect of cellular homeostasis and functioning. Proximity labeling mass spectrometry-based proteomics overcomes challenges typically associated with other methods and has quickly become the current state of the art in the field. Nevertheless, tight control of proximity-labeling enzymatic activity and expression levels is crucial to accurately identify protein interactors. Here, we leverage a T2A self-cleaving peptide and a non-cleaving mutant to accommodate the protein of interest in the experimental and control TurboID setup. To allow easy and streamlined plasmid assembly, we built a Golden Gate modular cloning system to generate plasmids for transient expression and stable integration. To highlight our T2A Split/link design, we applied it to identify protein interactions of the glucocorticoid receptor and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid and non-structural protein 7 (NSP7) proteins by TurboID proximity labeling. Our results demonstrate that our T2A split/link provides an opportune control that builds upon previously established control requirements in the field.
摘要：
蛋白质-蛋白质相互作用在细胞稳态和功能的各个方面都起着重要的生物学作用。基于邻近标记质谱的蛋白质组学克服了通常与其他方法相关的挑战，并迅速成为本领域的最新技术。然而，严格控制邻近标记的酶活性和表达水平对于准确识别蛋白质相互作用物至关重要。这里,我们利用T2A自切割肽和非切割突变体来适应实验和对照TurboID设置中的目的蛋白。为了方便和流线型的质粒组装，我们建立了一个金门模块化克隆系统来产生质粒，用于瞬时表达和稳定整合。为了突出我们的T2A拆分/链接设计，我们将其应用于通过TurboID邻近标记鉴定糖皮质激素受体与严重急性呼吸综合征冠状病毒2（SARS-CoV-2）核衣壳和非结构蛋白7（NSP7）蛋白的蛋白相互作用。我们的结果表明，我们的T2A拆分/链接提供了一个适当的控制，建立在先前在现场建立的控制要求。
