食品与发酵工业 >

2019 , Vol. 45 >Issue 16: 1 - 10

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.020730

研究报告

L-天冬氨酸-α-脱羧酶基因5′端二级结构对重组表达的抑制机制及消除策略

  • 令狐梅 ,
  • 韩来闯 ,
  • 周哲敏 ,
  • 崔文璟
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  • 1 (江南大学 生物工程学院,江苏 无锡,214122)
    2 (工业生物技术教育部重点实验室(江南大学),江苏 无锡,214122)
硕士研究生(崔文璟副教授为通讯作者,E-mail:wjcui@jiangnan.edu.cn)。

收稿日期: 2019-04-02

  网络出版日期: 2019-09-23

基金资助

国家自然科学基金-青年基金(31400058)

收起

Inhibition mechanism of recombinant expression by 5′terminal secondary structure of L-aspartate-α-decarboxylase gene and elimination strategy

  • LINGHU Mei ,
  • HAN Laichuang ,
  • ZHOU Zhemin ,
  • CUI Wenjing
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  • 1 (School of Biotechnology, Jiangnan University, Wuxi 214122, China)
    2 (Key Laboratory of Industrial Biotechnology, Ministry of Education(Jiangnan University), Wuxi 214122, China)

Received date: 2019-04-02

  Online published: 2019-09-23

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摘要

探究赤拟谷盗Tribolium castaneum L-天冬氨酸-α-脱羧酶(PanD)与人工高效表达元件适配过程中翻译抑制产生的机制,并发展新的消除策略。首先在枯草芽孢杆菌中利用高效表达元件验证翻译抑制现象并分析了翻译抑制产生的机制,同时融合sfGFP考察其对翻译抑制的影响,筛选与PanD酶适配性强的精简融合肽。结果显示,强组成型启动子不能介导PanD高表达,呈翻译抑制。分析证实,panD转录后mRNA的5′编码区与5′UTR形成抑制性的二级结构,降低翻译起始效率。将两种报告基因和其N端部分序列作为绝缘肽分别与PanD的N端融合后均能够提升重组蛋白的表达水平,消除由mRNA分子内部的顺式作用导致的翻译抑制现象。这为深入探索利用通用型肽绝缘子元件稳定异源基因在底盘中的表达提供依据。

关键词： L-天冬氨酸-α-脱羧酶; mRNA二级结构; N端截断; 融合肽; 枯草芽孢杆菌

本文引用格式

令狐梅 , 韩来闯 , 周哲敏 , 崔文璟 . L-天冬氨酸-α-脱羧酶基因5′端二级结构对重组表达的抑制机制及消除策略[J]. 食品与发酵工业, 2019 , 45(16) : 1 -10 . DOI: 10.13995/j.cnki.11-1802/ts.020730

Abstract

The purpose of this study was to explore the mechanism of translation inhibition of L-aspartate-α-decarboxylase (PanD) from Tribolium castaneum and develop a novel strategy to eliminate the inhibition. Firstly, the inhibited translation was verified by efficient expression elements, and the mechanism of inhibition was analyzed in Bacillus subtilis. Meanwhile, sfGFP was fused to investigate its effect on translation inhibition to screen reduced fusion peptides that had strong compatibility with PanD. The results showed that the strongly constitutive promoter could not mediate high expression of PanD and displayed translation inhibition. The analysis confirmed that the 5′ coding region and 5′UTR of panD post-transcriptional mRNA formed an inhibitory secondary structure and reduced the initiation efficiency of translation. Moreover, when the two reporter genes and part of the N-terminal sequence of the reporter gene were used as insulating peptides to fuse with the N-terminal of PanD in individual, the expression level of the recombinant protein could be improved, and the translational inhibition caused by the cis-acting effect within the mRNA molecule was eliminated. Overall, this study provides a basis to systematically design a universal peptide insulator to stabilize gene expression in microbial synthetic chassis.

Key words： L-aspartate-α-decarboxylase; secondary structure of mRNA; N-terminal truncation; fusion peptide; Bacillus subtilis

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