荧光非天然氨基酸的遗传编码作为 FRET 供体用于去泛素化酶活性分析。
Genetic Encoding of a Fluorescent Noncanonical Amino Acid as a FRET Donor for the Analysis of Deubiquitinase Activities.
机构信息
State Key Laboratory of Chemical Oncogenomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen, China.
Institute of Chemical Biology, Shenzhen Bay Laboratory, Shenzhen, China.
出版信息
Methods Mol Biol. 2023;2676:55-67. doi: 10.1007/978-1-0716-3251-2_4.
The genetic code expansion technology enables the genetic encoding of fluorescent noncanonical amino acids (ncAAs) for site-specific fluorescent labeling of proteins. These co-translational and internal fluorescent tags have been harnessed to establish genetically encoded Förster resonance energy transfer (FRET) probes for studying protein structural changes and interactions. Here, we describe the protocols for site-specific incorporation of an aminocoumarin-derived fluorescent ncAA into proteins in E. coli and preparation of a fluorescent ncAA-based FRET probe for assaying the activities of deubiquitinases, a key class of enzymes involved in ubiquitination. We also describe the deployment of an in vitro fluorescence assay to screen and analyze small-molecule inhibitors against deubiquitinases.
遗传密码扩展技术使遗传编码荧光非天然氨基酸（ncAA）成为可能，从而实现蛋白质的定点荧光标记。这些共翻译和内部荧光标签已被用于建立遗传编码的Förster 共振能量转移（FRET）探针，用于研究蛋白质结构变化和相互作用。在这里，我们描述了在大肠杆菌中定点掺入氨基香豆素衍生的荧光 ncAA 的方法，并制备了基于荧光 ncAA 的 FRET 探针，用于测定去泛素化酶的活性，去泛素化酶是参与泛素化的关键酶类之一。我们还描述了体外荧光测定法的应用，用于筛选和分析针对去泛素化酶的小分子抑制剂。
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