Biochemical isolation of Argonaute protein complexes by Ago-APP
Biochemical isolation of Argonaute protein complexes by Ago-APP
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DOI：
10.1073/pnas.1506116112
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发表时间：
2015-09-22
影响因子：
11.1
通讯作者：
Meister, Gunter
中科院分区：
文献类型：
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作者：
Hauptmann, Judith;Schraivogel, Daniel;Meister, Gunter
在 microRNA (miRNA) 引导的基因沉默过程中，Argonaute (Ago) 蛋白与 TNRC6/GW 蛋白家族的成员相互作用。在这里，我们使用了与 GST 融合的短 GW 蛋白衍生肽，并证明它以高亲和力与 Ago 蛋白结合。这样可以同时分离不同物种中表达的所有 Ago 蛋白复合物，以鉴定相关蛋白、小 RNA 或目标 mRNA。我们将我们的方法称为“Ago 肽亲和纯化”(Ago-APP)。此外，该肽的表达会竞争内源性 TNRC6 蛋白，导致哺乳动物细胞中 miRNA 功能的全面抑制。
During microRNA (miRNA)-guided gene silencing, Argonaute (Ago) proteins interact with a member of the TNRC6/GW protein family. Here we used a short GW protein-derived peptide fused to GST and demonstrate that it binds to Ago proteins with high affinity. This allows for the simultaneous isolation of all Ago protein complexes expressed in diverse species to identify associated proteins, small RNAs, or target mRNAs. We refer to our method as "Ago protein Affinity Purification by Peptides" (Ago-APP). Furthermore, expression of this peptide competes for endogenous TNRC6 proteins, leading to global inhibition of miRNA function in mammalian cells.