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在高温下开发热友宿主-载体系统,用于在高温下生产重组蛋白质
Ryota Kurashiki1, Kosuke Koyama1, Yukina Sakaguchi2
1Department of Engineering, Graduate School of Sustainability Science, Tottori University, 4-101 Koyama-Minami, Tottori, 680-8552, Japan.
Applied microbiology and biotechnology
|September 27, 2023
概括
使用Geobacillus物种的新热友系统有效地产生重组蛋白质. 该系统利用pGKE124等离子体上修饰的促进剂,实现了高产量,包括1g/L的细胞外酶.
科学领域:
- 生物技术是生物技术.
- 微生物工程 微生物工程
- 蛋白质生产 蛋白质生产
背景情况:
- 地基菌种是热爱动物,以高效的重组蛋白质生产而闻名.
- 在Geobacillus kaustophilus HTA426中识别强大的促进体对于优化蛋白质产量至关重要.
- 转录组数据表明,在繁殖阶段基因表达高,这表明潜在的促进者候选人.
研究的目的:
- 在Geobacillus kaustophilus中表征强大的促进剂,以增强重组蛋白质生产.
- 开发一个高效的宿主-载体系统,以在高温下高水平的蛋白质表达.
- 设计一种稳定的等离子体载体,用于热友性Geobacillus物种.
主要方法:
- 使用金星光蛋白 (VFP) 的记者测定被用来描述60°C的促进体活性.
- 一个突变的cspD促进体 (PcspD) 被设计成在Geobacillus中有效运作,但在Escherichia coli中没有.
- 使用突变的促进子来克隆和表达热爱动物,构建了pGKE124等离子体.
主要成果:
- 本地cspD促进体 (PcspD) 在60°C时在G. kaustophilus中显示出强大的VFP表达.
- 一种突变的PcspD使大肠杆菌成功克隆和高VFP产量 (390毫克/升) 在G. kaustophilus.
- 在其他热友物种中,pGKE124系统实现了高产量,特别是G. thermodenitrificans K1041,在1g/L的细胞外酶生产中.
结论:
- 一个新的热友宿主-载体系统已成功构建用于重组蛋白质生产.
- 该系统表现出多功能性,在高温下以廉价的介质生产各种蛋白质.
- 开发的系统为高产量的细胞外蛋白质生产提供了一个有效的平台,以克西兰酶为例.
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