{"title":"利用靶向芽殖酵母中 GFP 或 mCherry 的纳米抗体,一步生成基于 AlissAID 的条件性基因敲除菌株。","authors":"Yoshitaka Ogawa, Taisei P Ueda, Keisuke Obara, Kohei Nishimura, Takumi Kamura","doi":"10.21769/BioProtoc.5019","DOIUrl":null,"url":null,"abstract":"<p><p>The Auxin-inducible degron (AID) system is a genetic tool that induces rapid target protein depletion in an auxin-dependent manner. Recently, two advanced AID systems-the super-sensitive AID and AID 2-were developed using an improved pair of synthetic auxins and mutated TIR1 proteins. In these AID systems, a nanomolar concentration of synthetic auxins is sufficient as a degradation inducer for target proteins. However, despite these advancements, AID systems still require the fusion of an AID tag to the target protein for degradation, potentially affecting its function and stability. To address this limitation, we developed an affinity linker-based super-sensitive AID (AlissAID) system using a single peptide antibody known as a nanobody. In this system, the degradation of GFP- or mCherry-tagged target proteins is induced in a synthetic auxin (5-Ad-IAA)-dependent manner. Here, we introduce a simple method for generating AlissAID strains targeting GFP or mCherry fusion proteins in budding yeasts. Key features • AlissAID system enables efficient degradation of the GFP or mCherry fusion proteins in a 5-Ad-IAA-depending manner. • Transforming the pAlissAID plasmids into strains with GFP- or mCherry- tagged proteins.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0000,"publicationDate":"2024-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11211076/pdf/","citationCount":"0","resultStr":"{\"title\":\"A Single-step Generation of AlissAID-based Conditional Knockdown Strains Using Nanobody that Targets GFP or mCherry in Budding Yeast.\",\"authors\":\"Yoshitaka Ogawa, Taisei P Ueda, Keisuke Obara, Kohei Nishimura, Takumi Kamura\",\"doi\":\"10.21769/BioProtoc.5019\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>The Auxin-inducible degron (AID) system is a genetic tool that induces rapid target protein depletion in an auxin-dependent manner. Recently, two advanced AID systems-the super-sensitive AID and AID 2-were developed using an improved pair of synthetic auxins and mutated TIR1 proteins. In these AID systems, a nanomolar concentration of synthetic auxins is sufficient as a degradation inducer for target proteins. However, despite these advancements, AID systems still require the fusion of an AID tag to the target protein for degradation, potentially affecting its function and stability. To address this limitation, we developed an affinity linker-based super-sensitive AID (AlissAID) system using a single peptide antibody known as a nanobody. In this system, the degradation of GFP- or mCherry-tagged target proteins is induced in a synthetic auxin (5-Ad-IAA)-dependent manner. Here, we introduce a simple method for generating AlissAID strains targeting GFP or mCherry fusion proteins in budding yeasts. Key features • AlissAID system enables efficient degradation of the GFP or mCherry fusion proteins in a 5-Ad-IAA-depending manner. • Transforming the pAlissAID plasmids into strains with GFP- or mCherry- tagged proteins.</p>\",\"PeriodicalId\":93907,\"journal\":{\"name\":\"Bio-protocol\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.0000,\"publicationDate\":\"2024-06-20\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11211076/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Bio-protocol\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.21769/BioProtoc.5019\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Bio-protocol","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.21769/BioProtoc.5019","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOLOGY","Score":null,"Total":0}
引用次数: 0
摘要
辅助素诱导降解子(AID)系统是一种遗传工具,能以辅助素依赖的方式诱导目标蛋白的快速消耗。最近,利用一对改进的合成辅助素和突变的 TIR1 蛋白,开发出了两种先进的 AID 系统--超灵敏 AID 和 AID 2。在这些 AID 系统中,纳摩尔浓度的合成助剂就足以诱导目标蛋白质降解。然而,尽管取得了这些进步,AID 系统仍然需要将 AID 标签与目标蛋白融合才能降解,这可能会影响目标蛋白的功能和稳定性。为了解决这一局限性,我们开发了一种基于亲和连接体的超灵敏 AID(AlissAID)系统,使用的是一种称为纳米抗体的单肽抗体。在该系统中,GFP 或 mCherry 标记的目标蛋白的降解是以合成辅助素(5-Ad-IAA)依赖的方式诱导的。在这里,我们介绍了一种在芽殖酵母中生成靶向 GFP 或 mCherry 融合蛋白的 AlissAID 菌株的简单方法。主要特点 - AlissAID 系统能以 5-Ad-IAA 依赖性方式高效降解 GFP 或 mCherry 融合蛋白。- 将 pAlissAID 质粒转化为带有 GFP 或 mCherry 标记蛋白的菌株。
A Single-step Generation of AlissAID-based Conditional Knockdown Strains Using Nanobody that Targets GFP or mCherry in Budding Yeast.
The Auxin-inducible degron (AID) system is a genetic tool that induces rapid target protein depletion in an auxin-dependent manner. Recently, two advanced AID systems-the super-sensitive AID and AID 2-were developed using an improved pair of synthetic auxins and mutated TIR1 proteins. In these AID systems, a nanomolar concentration of synthetic auxins is sufficient as a degradation inducer for target proteins. However, despite these advancements, AID systems still require the fusion of an AID tag to the target protein for degradation, potentially affecting its function and stability. To address this limitation, we developed an affinity linker-based super-sensitive AID (AlissAID) system using a single peptide antibody known as a nanobody. In this system, the degradation of GFP- or mCherry-tagged target proteins is induced in a synthetic auxin (5-Ad-IAA)-dependent manner. Here, we introduce a simple method for generating AlissAID strains targeting GFP or mCherry fusion proteins in budding yeasts. Key features • AlissAID system enables efficient degradation of the GFP or mCherry fusion proteins in a 5-Ad-IAA-depending manner. • Transforming the pAlissAID plasmids into strains with GFP- or mCherry- tagged proteins.