利用基于细胞的筛选来确定适合生物降解的 E3 连接酶的方案。

IF 1.3 Q4 BIOCHEMICAL RESEARCH METHODS

STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-10-24 DOI:10.1016/j.xpro.2024.103413

Marie Sorbara, Margot Cristol, Anaïs Cornebois, Klervi Desrumeaux, Pierre Cordelier, Nicolas Bery

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引用次数: 0

摘要

在这里，我们提供了一种方案，利用基于细胞的筛选试验，鉴定作为生物降解物时具有功能性的 E3 泛素连接酶。我们描述了建立表达 GFP 标记的感兴趣蛋白质（POI）的稳定细胞系、准备要筛选的 E3 连接酶子库以及进行细胞筛选的步骤。该方案可广泛应用于生物降解环境中任何功能性 E3 连接酶的鉴定。有关使用和执行该方案的完整细节，请参阅 Cornebois 等人的文章1。

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Protocol to identify E3 ligases amenable to biodegraders using a cell-based screening.

Here, we provide a protocol for the identification of E3 ubiquitin ligases that are functional when implemented as biodegraders using a cell-based screening assay. We describe steps for establishing a stable cell line expressing a GFP-tagged protein of interest (POI), preparing a sub-library of E3 ligases to screen, and performing the cell-based screening. This protocol can be broadly applied to identify any functional E3 ligase in a biodegrader setting. For complete details on the use and execution of this protocol, please refer to Cornebois et al.¹.

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