通过in vivo高突变,对氨基酸-tRNA合成酶的定向进化
bioRxiv : the preprint server for biology
|October 10, 2024
概括
遗传密码扩展 (GCE) 使用工程氨基-tRNA合成酶 (aaRS) /tRNA对将非正规氨基酸 (ncAA) 纳入蛋白质. 一个基于OrthoRep的新策略快速开发出高效的aaRSs,用于更广泛的GCE应用.
科学领域:
- 合成生物学 合成生物学
- 分子生物学分子生物学
- 生物化学 生化学
背景情况:
- 遗传密码扩展 (GCE) 允许使用正交氨基酸-tRNA合成酶 (aaRS) /tRNA对进行非正规氨基酸 (ncAA) 的特定位点内置.
- 发展新的aaRS/tRNA对的传统方法是劳动密集型的,并产生低于最佳的整合效率.
- 有效的aaRS/tRNA对对于扩大GCE在生物研究和生物技术中的实用性至关重要.
研究的目的:
- 开发和演示一种新的策略,用于在酵母中使用OrthoRep介导的连续超突变来演变氨基-tRNA合成酶 (aaRSs).
- 通过OrthoRep平台开发的aaRSs来评估ncAA整合的效率和范围.
- 探索这个平台的潜力,以加速开发用于遗传密码扩展的新工具.
主要方法:
- 采用OrthoRep系统来驱动酵母菌中现有的氨基酸-tRNA合成酶 (aaRS) 基因的持续高突变.
- 进行了8个独立的进化运动,使用了4个不同的aaRS/tRNA亲基,针对7个不同的非正规氨基酸 (ncAA).
- 量化了ncAA纳入蛋白质的效率,以应对珀停止码.
主要成果:
- 成功进化了多个新型aaRSs,能够通过珀色子抑制将一系列13种已测试的ncAA纳入蛋白质.
- 在一些进化系统中,实现了 ncAA 整合效率,与从酵母中的感觉编码子转化到天然氨基酸的效率相当.
- 发现了一种aaRS变体,它进化了其表达的自我调节,增加了对ncAAs的翻译依赖.
结论:
- 由OrthoRep驱动的aaRS进化策略显著加速了用于遗传密码扩展的高效工具的开发.
- 该平台能够快速生成具有广泛ncAA兼容性和高整合效率的新型aaRS.
- 这些发现突显了OrthoRep在推进GCE技术和扩大其在合成生物学中的应用方面的潜力.
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