生物学杂志 ›› 2022, Vol. 39 ›› Issue (6): 101-.doi: 10.3969/j.issn.2095-1736.2022.06.101

• 技术方法 • 上一篇    下一篇

重叠基因在异源蛋白表达中的新应用

  

  1. 1. 天津科技大学 生物工程学院, 天津 300457; 2. 中国科学院天津工业生物技术研究所
    系统微生物工程重点实验室, 天津 300308
  • 出版日期:2022-12-18 发布日期:2022-12-12
  • 通讯作者: 张娟琨,博士,教授,研究方向为酶生物传感器,E-mail: zhangjk@tust.edu.cn;江会锋,博士,研究员,研究方向为合成生物学,E-mail: jiang_hf@tib.cas.cn
  • 作者简介:彭凯,硕士研究生,研究方向为蛋白表达调控,E-mail: pengk@tib.cas.cn
  • 基金资助:
    国家自然科学基金项目(31901015)

New application of overlapping genes in the expression of heterologous proteins

  1. 1. School of Biological Engineering, Tianjin University of Science and Technology, Tianjin 300457, China;
    2. Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology,
    Chinese Academy of Sciences, Tianjin 300308, China
  • Online:2022-12-18 Published:2022-12-12

摘要: 大肠杆菌在调控异源蛋白的表达量和可溶性方面仍然存在困难。研究开发一种融合重叠基因结构和mRNA 5′-端序列优化的蛋白表达调控方法。在表达基因起始密码子前引入两个碱基TG,利用pET-28a质粒载体表达框的5′-端标签,与其形成TGATG的重叠基因类型的表达结构。再通过设计简并引物对异源基因mRNA的5′-端连续14个密码子区域进行同义密码子突变,改变mRNA的二级结构,实现重叠基因结构强度筛选。利用这种方法,在不改变质粒组成和外界条件的情况下就能影响异源蛋白的表达量,并通过影响翻译过程速率来调控蛋白可溶性。通过对多个异源基因表达优化实例,证明重叠基因结构在调控蛋白可溶性表达中的作用。为大肠杆菌中异源蛋白在表达量和可溶性问题的优化方面提供一种简单的调控策略。

关键词: 大肠杆菌, 异源表达, 同义密码子, mRNA结构, 重叠基因

Abstract: There are still difficulties in regulating the expression and solubility of heterologous proteins in E. coli. In this study, a protein expression optimization method that combines overlapping gene structure and 5′-end sequence optimization of mRNA was developed. Utilizing the 5′-end tag of the pET-28a plasmid vector expression box, and the target gene introduced into TG before the start codon formed an overlapping gene type expression structure of TGATG. Then, by designing degenerate primers, synonymous codon mutations of 14 consecutive codons at the 5′-end of a heterologous gene mRNA were performed to change the secondary structure of mRNA and realize the screening of overlapping gene structure strength. This method can affect the expression of heterologous protein without changing the plasmid composition and external conditions, and regulate the solubility of the protein by affecting the rate of the translation process. Several examples of heterologous gene expression optimization in this study have proved the role of overlapping gene structures in regulating the soluble expression of proteins. It provided a simple optimization strategy for the regulation of the expression and solubility of heterologous proteins in E. coli.

Key words: E. coli, heterologous expression, synonymous codon, mRNA structure, overlapping genes

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