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mRNA初始区域对蛋白质生产的影响:使用重组排毒肺清素作为模型的案例研究

Filipe Fusco^1,2, Manuella Cazelato Pires^1,2, Alexandre Paulo Yague Lopes¹

¹Laboratory of Vaccine Development, Butantan Institute, Sao Paulo, Brazil.

Frontiers in bioengineering and biotechnology

|January 23, 2024

概括

优化重组蛋白质生产包括分析5'mRNA二次结构. 修改这个区域,比如添加His-tag或改变基因序列,通过提高翻译效率来显著提高蛋白质产量.

科学领域:

生物技术是生物技术.
分子生物学分子生物学
生物工艺工程生物工艺工程

背景情况:

重组蛋白质生产对于生物制药至关重要,但面临着低产量的挑战.
优化努力传统上集中在生物反应器和净化过程上,往往忽视生物分子方面的方面.
5'mRNA二次结构在翻译效率和蛋白质产量的作用经常被低估.

研究的目的:

研究5'mRNA二次结构对重组蛋白质生产的影响.
评估N-终端His-tag和同义核酸变化对翻译效率的影响.
为了证明in silico mRNA分析在优化蛋白质产量的实用性.

主要方法:

培养大肠杆菌BL21(DE3) 克隆,在10L生物反应器中产生复合性排毒肺清素 (PdT).
与pdt基因的两个不同版本的PdT生产与没有N终端的His-tag和pdt基因的比较.
使用TIsigner和RNAfold进行mRNA二次结构的in silico分析.

主要成果:

存在一个N端的His标签,至少增加了1.5倍的目标蛋白质合成.
在分析中发现,His-tag创建了一个mRNA二级结构,更容易翻译.
经过修改的pdt基因 (版本2) 具有同义变化,其mRNA开放能量较低,即使没有标签,PdT的产生也更高.

结论:

5'mRNA二次结构显著影响重组蛋白质生产产量.
简单的in silico mRNA分析可以指导增强蛋白位的基因设计.
将mRNA结构分析集成到异质基因设计中,与传统的生物处理策略相比,可以加速优化.

关键词:

他的标签是他的标签.在 PdTT 的情况下.肺炎链球菌的肺炎链球菌.生物反应器是一个生物反应器.在分析中,分析.mRNA二次结构的mRNA二次结构再组合的大肠杆菌埃舍里奇亚开始翻译开始翻译

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