一种灵活的"插即用"双结构及其在对大肠杆菌蛋白质表达系统的查中的应用
Binbin Feng1, Weiwei Hu1, Yingyi Duan1
1Microbial and Viral Platform, WuXi Biologics (Hangzhou FTZ) Co., Ltd, #9, 388 of No.16 street, Baiyang Avenue, Hangzhou Free Trade Zone, Hangzhou, Zhejiang Province, China.
Journal of microbiological methods
|April 7, 2024
概括
这项研究引入了一种新的二基斯特朗表达系统,用于量化大肠杆菌 (E. coli) 中的排序酶生成. 该系统使用mCherry光报告器进行准确的,非侵入性的蛋白质标位测量.
科学领域:
- 生物技术是生物技术.
- 分子生物学分子生物学
- 蛋白质表达方式
背景情况:
- 带有光报道器的双晶体表达系统为检测蛋白质水平的凝电泳等传统方法提供了更简单的替代方案.
- 现有的系统往往缺乏定量准确性,原因是表达滞后和对蛋白的负面影响.
研究的目的:
- 开发一个插即用的双结构,用于对大肠杆菌的定量排序酶表达查.
- 为了建立光记者强度和蛋白标位之间的可靠相关性.
主要方法:
- 设计了一种使用mCherry作为光记者的新型双晶构造.
- 该构造使报告员和Sortase在单个推动者下共同转录开放阅读框架 (ORF).
- 该系统用于选E. coli中Sortase表达的最佳复制体和促进体.
主要成果:
- 在Sortase表达位和mCherry光强度之间观察到强烈的正定量相关性 (R2>0.97).
- 报告者基因表达没有对目标蛋白质产量产生显著的负面影响.
- 建立了一种定量模型,用于非侵入性测量标蛋白位.
结论:
- 开发的插电式双电子系统能够准确,非侵入性量化大肠杆菌中Sortase表达的E. coli.
- 该系统有助于有效选表达条件,并可以通过去除记者基因来适应工业应用.
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