Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
Central Laboratory, Changzhi Medical College, Changzhi 046000, China.
Biomolecules. 2021 Mar 31;11(4):524. doi: 10.3390/biom11040524.
The prokaryotic Argonaute proteins (pAgos) have been reported to cleave or interfere with DNA targets in a guide-dependent or independent manner. It is often difficult to characterize pAgos in vivo due to the extreme environments favored by their hosts. In the present study, we expressed functional pAgo (TtAgo) in BL21 (DE3) cells at 37 °C. Initial attempts to express TtAgo in BL21(DE3) cells at 37 °C failed. This was not because of TtAgo mediated general toxicity to the host cells, but instead because of TtAgo-induced loss of its expression plasmid. We employed this discovery to establish a screening system for isolating loss-of-function mutants of TtAgo. The gene was used to help select for full-length TtAgo loss of function mutants, as overexpression of renders the cell to be resistant to the triclosan. We isolated and characterized eight mutations in TtAgo that abrogated function. The ability of TtAgo to induce loss of its expression vector in vivo at 37 °C is an unreported function that is mechanistically different from its reported in vitro activity. These results shed light on the mechanisms by which TtAgo functions as a defense against foreign DNA invasion.
原核 Argonaute 蛋白（pAgos）已被报道以依赖于指导或独立于指导的方式切割或干扰 DNA 靶标。由于其宿主偏好的极端环境，通常难以在体内表征 pAgos。在本研究中，我们在 37°C 的 BL21（DE3）细胞中表达了功能性 pAgo（TtAgo）。最初试图在 37°C 的 BL21（DE3）细胞中表达 TtAgo 失败了。这不是因为 TtAgo 介导的对宿主细胞的普遍毒性，而是因为 TtAgo 诱导其表达质粒的丢失。我们利用这一发现建立了一个筛选系统，用于分离 TtAgo 的功能丧失突变体。该基因用于帮助选择全长 TtAgo 功能丧失突变体，因为 基因的过表达使细胞对三氯生具有抗性。我们分离并鉴定了 TtAgo 中的八个导致功能丧失的突变。TtAgo 在 37°C 下体内诱导其表达载体丢失的能力是一种未报道的功能，其机制与体外报道的活性不同。这些结果揭示了 TtAgo 作为抵御外来 DNA 入侵的防御机制。
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