Food and Fermentation Industries >

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

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

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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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

Cite this article

LINGHU Mei , HAN Laichuang , ZHOU Zhemin , CUI Wenjing . Inhibition mechanism of recombinant expression by 5′terminal secondary structure of L-aspartate-α-decarboxylase gene and elimination strategy[J]. Food and Fermentation Industries, 2019 , 45(16) : 1 -10 . DOI: 10.13995/j.cnki.11-1802/ts.020730

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