Targeted Protein Depletion Using the Auxin‐Inducible Degron 2 (AID2) System
Targeted Protein Depletion Using the Auxin‐Inducible Degron 2 (AID2) System
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DOI：
10.1002/cpz1.219
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发表时间：
2021-08
期刊：
影响因子：
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通讯作者：
Yuichiro Saito;Masato T. Kanemaki
中科院分区：
文献类型：
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作者：
Yuichiro Saito;Masato T. Kanemaki
使用条件降解决定子的靶向蛋白质消耗是探测蛋白质在活细胞中的作用的有力方法，因为消耗可以被诱导的速度及其可逆性。生长素诱导降解决定子（AID）是细胞生物学中最常见的基于降解决定子的技术之一。我们最近建立了一个改进的系统，称为AID 2，它涉及表达突变E3连接酶亚基OsTIR 1（F74 G），并将感兴趣的蛋白质融合到mini-AID（mAID）标签上，并采用了一种新的更有效的配体5-苯基-吲哚-3-乙酸（5-Ph-IAA）。AID 2系统克服了与原始AID系统相关联的一些缺点，即，没有生长素的渗漏降解和高生长素剂量的需求。因此，使用AID 2，现在可以更精确地控制降解决定子融合蛋白，通过添加低剂量的5-Ph-IAA，使靶蛋白在10至45分钟的半衰期内降解。重要的是，在AID 2中，不需要控制OsTIR 1（F74 G）的表达来抑制渗漏降解，并且组成型表达OsTIR 1（F74 G）的亲本细胞系可以用于产生多种mAID标记的蛋白质。在这里，我们描述了一个协议，标记的内源性蛋白质与mAID在二倍体HCT 116细胞。我们的协议可以适用于其他哺乳动物细胞系，并将提高艾滋病2研究活细胞中的蛋白质功能的效用。© 2021 Wiley Periodicals LLC.
Targeted protein depletion using a conditional degron is a powerful method to probe the role of proteins in living cells because of the speed with which depletion can be induced and its reversibility. The auxin‐inducible degron (AID) is one of the most common degron‐based technologies used in cell biology. We recently established an improved system, called AID2, which involves expressing a mutant E3 ligase subunit, OsTIR1(F74G), and fusing a protein of interest to the mini‐AID (mAID) tag, and that employs a new and more potent ligand, 5‐phenyl‐indole‐3‐acetic acid (5‐Ph‐IAA). The AID2 system overcomes some of the drawbacks associated with the original AID system, i.e., leaky degradation without auxin and the requirement of high auxin doses. With AID2 it is, therefore, now possible to control a degron‐fused protein more precisely, enabling target proteins to be degraded with a half‐life of 10 to 45 min via the addition of a low dose of 5‐Ph‐IAA. Importantly, in AID2, it is not necessary to control the expression of OsTIR1(F74G) for suppressing leaky degradation and a parental cell line constitutively expressing OsTIR1(F74G) can be used for the generation of multiple mAID‐tagged proteins. Here, we describe a protocol for the tagging of endogenous proteins with mAID in diploid HCT116 cells. Our protocol can be applied to other mammalian cell lines and will enhance the utility of AID2 for studying protein functions in living cells. © 2021 Wiley Periodicals LLC.