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, Elizabeth C Gardner3,2, 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.
iScience
|January 15, 2025
概括
科学家们设计了一种新的T7RNA聚合酶 (RNAP) 酶,该酶可以在转录中添加必要的5'盖子. 这一突破显著提高了真核生物系统中的蛋白质生产和基因调节.
科学领域:
- 合成生物学 合成生物学
- 分子生物学分子生物学
- 生物技术是生物技术.
背景情况:
- T7RNA聚合酶 (RNAP) 广泛用于原核生物的基因表达.
- 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,阻碍了效率.
- 正角基因表达系统对于先进的合成生物学应用至关重要.
研究的目的:
- 开发一个改进的T7RNAP系统用于真核生物基因表达.
- 在真核宿主中克服T7RNAP的限制缺陷.
- 为合成生物学创建一个强大而通用的直角基因调节系统.
主要方法:
- 进化了一个融合酶,将T7 RNAP与非洲猪瘟病毒封闭酶结合起来.
- 使用 *Saccharomyces cerevisiae* (酵母) 进行酶进化.
- 在酵母和哺乳动物细胞中测试了工程变体,用于蛋白质表达和基因电路功能.
主要成果:
- 孤立的高度活跃的融合酶变体.
- 达到的蛋白质表达水平大约是比野生类型T7 RNAP高两倍.
- 在酵母中使用基于T7RNAP的基因电路来证明可编程的基因表达控制.
- 在哺乳动物细胞中得到验证的增强性能.
结论:
- 工程融合酶为真核生物提供了一个强大的,直角的基因调节系统.
- 这个系统显著提高了蛋白质表达和基因调节效率.
- 开发的技术扩大了T7 RNAP在多种真核生物合成生物学应用中的实用性.
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