Generation of conditional auxin-inducible degron (AID) cells and tight control of degron-fused proteins using the degradation inhibitor auxinole
Generation of conditional auxin-inducible degron (AID) cells and tight control of degron-fused proteins using the degradation inhibitor auxinole
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DOI：
10.1016/j.ymeth.2019.04.010
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发表时间：
2019-07-01
期刊：
影响因子：
4.8
通讯作者：
Kanemaki, Masato T.
中科院分区：
文献类型：
--
作者：
Yesbolatova, Aisha;Natsume, Toyoaki;Kanemaki, Masato T.
使用降解决定子控制蛋白质表达是有利的，因为感兴趣的蛋白质可以以可逆的方式快速耗尽。我们通过将植物特异性降解途径移植到非植物细胞中，开创了生长素诱导降解决定子（AID）技术的发展。在表达E3连接酶组分OsTIR 1的人细胞中，在植物激素生长素的存在下，可以降解半衰期为15-45分钟的脱克隆融合蛋白。我们先前报道了人HCT 116突变体的产生，其中内源性蛋白质的C末端通过基于CRISPR Cas9的敲入与降解决定子融合。在这里，我们展示了新的质粒的N-末端标记，并描述了一个详细的协议，用于产生艾滋病突变体的人HCT 116和DLD 1细胞。此外，我们报告使用OsTIR 1抑制剂，生长素，以抑制degron融合蛋白的泄漏降解。生长素的添加也可用于在耗尽degron融合蛋白后的快速再表达。这些改进增强了AID技术在研究活人类细胞中蛋白质功能方面的实用性。
Controlling protein expression using a degron is advantageous because the protein of interest can be rapidly depleted in a reversible manner. We pioneered the development of the auxin-inducible degron (AID) technology by transplanting a plant-specific degradation pathway to non-plant cells. In human cells expressing an E3 ligase component, OsTIR1, it is possible to degrade a degron-fused protein with a half-life of 15-45 min in the presence of the phytohormone auxin. We reported previously the generation of human HCT116 mutants in which the C terminus of endogenous proteins was fused with the degron by CRISPR Cas9-based knock-in. Here, we show new plasmids for N-terminal tagging and describe a detailed protocol for the generation of AID mutants of human HCT116 and DLD1 cells. Moreover, we report the use of an OsTIR1 inhibitor, auxinole, to suppress leaky degradation of degron-fused proteins. The addition of auxinole is also useful for rapid re-expression after depletion of degron-fused proteins. These improvements enhance the utility of AID technology for studying protein function in living human cells.