在真核生物中用于可编程基因表达的直角转录引擎的定向进化
Shaunak Kar1,2,3, Elizabeth C Gardner4,2,3, Kamyab Javanmardi2
1Laboratory of Antibody Discovery and Accelerated Protein Therapeutics, Center for Infectious Diseases, Houston Methodist Research Institute and Department of Pathology and Genomic Medicine, Houston Methodist Hospital, Houston, TX, USA.
bioRxiv : the preprint server for biology
|October 10, 2024
概括
研究人员设计了一种T7RNA聚合酶 (RNAP) 融合酶,并将其与用于真核生物基因表达的封闭酶结合起来. 这种增强的系统显著提高了蛋白质的生产,并提供了酵母和哺乳动物细胞的正交控制.
科学领域:
- 合成生物学 合成生物学
- 分子生物学分子生物学
- 基因表达规范 基因表达规范
背景情况:
- T7RNA聚合酶 (RNAP) 是一个强大的工具,用于基因表达和蛋白质的生产在核细胞.
- 在真核生物中,T7 RNAP的一个关键限制是转录物上缺乏5'甲基瓜诺辛盖,这阻碍了它的效用.
- 现有的T7RNAP系统在真核生物系统中缺乏广泛的适用性和效率.
研究的目的:
- 为真核宿主开发一个正交的基因调节系统.
- 通过结合一个限制机制来提高T7RNAP的效率.
- 在真核生物系统中改善重组蛋白质的生产.
主要方法:
- 进化了一个融合酶,将T7 RNAP与Saccharomyces cerevisiae中的非洲猪瘟病毒封闭酶结合起来.
- 工程融合酶的高度活性变体被隔离和特征化.
- 在酵母和哺乳动物细胞中使用基于T7RNAP的遗传电路来证明可编程的基因表达控制.
主要成果:
- 与野生型T7RNAP相比,工程融合酶变体显示出大约两倍的蛋白质表达.
- 在酵母中成功展示了可编程的基因表达控制.
- 在哺乳动物细胞中验证了工程变异的增强性能.
结论:
- 为各种真核宿主开发了一个强大且正交的基因调节系统.
- 改造的T7 RNAP融合酶显著提高了蛋白质表达和基因调节效率.
- 这个系统扩大了合成生物学在真核生物系统中的多功能性和适用性.
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