在Clostridium saccharoperbutylacetonicum中开发一种高效的重组蛋白表达系统,基于菌体T7系统
Yuechao Ma1,2, Na Guo1,2, Xiao Li3
1Department of Biosystems Engineering, Auburn University, Auburn, Alabama 36849, United States.
ACS synthetic biology
|September 15, 2023
概括
为无氧宿主开发了一种新的T7菌体表达系统,使Clostridium saccharoperbutylacetonicum中能够产生高水平的重组蛋白质. 这一突破促进了工业生物技术的大规模无内毒素蛋白质制造.
科学领域:
- 生物技术是生物技术.
- 分子生物学分子生物学
- 微生物工程 微生物工程
背景情况:
- 重组蛋白质生产至关重要,但缺乏对大规模无氧宿主有效的系统.
- 严格的无氧细菌,如Clostridium物种,对于工业应用是有价值的,但对异质蛋白质表达具有挑战性.
研究的目的:
- 在严格无氧宿主中开发和优化一种新的,严格规范的,可诱导的重组蛋白表达系统.
- 在Clostridium saccharoperbutylacetonicum中使用菌体T7系统建立一个强大的平台,用于高水平的异质蛋白的生产.
主要方法:
- 通过将编码子优化的T7RNA聚合酶基因整合到染色体中,设计了一个Clostridium saccharoperbutylacetonicum菌株.
- 利用一个单一的等离子体与T7促进体,由乳糖诱导系统调节,用于正交基因表达.
- 通过删除β-galactosidase基因优化了系统,并评估了对内源性促进体的转录强度.
主要成果:
- 与强大的内源性促进剂相比,在优化菌株 (TM-07) 中,T7促进剂的转录强度增加了9.5倍.
- 成功表达了一种异质酶,NADP+依赖的3-基基甲酸脱酶 (Hbd1),达到总细胞蛋白质的30.4-42.4%.
- 在原始和纯化的酶制剂中显著增强了酶活性 (比原始活性高8.3倍).
结论:
- 开发的基于T7的表达系统是第一个在Clostridium物种中应用的,提供严格的控制和高水平的蛋白质生产.
- 该系统使得在严格无氧条件下高效,大规模,无内毒素的重组蛋白质生产成为可能.
- 优化的系统具有显著的潜力,可以促进生物技术和工业应用,这些应用需要无氧蛋白质制造.
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