Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Elucidating Bottlenecks to the Efficient Preparation of AB5-Hexamer Mucosal Adjuvant Protein LTm by Genetic Engineering

  • Liu, Di (School of Biotechnology and State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology) ;
  • Hu, Fabiao (School of Biotechnology and State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology) ;
  • Wang, Wenpeng (School of Biotechnology and State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology) ;
  • Wu, Dong (School of Biotechnology and State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology) ;
  • He, Xiujuan (School of Pharmacy, Shanghai Key Laboratory of New Drug Design, East China University of Science and Technology) ;
  • Zheng, Wenyun (School of Pharmacy, Shanghai Key Laboratory of New Drug Design, East China University of Science and Technology) ;
  • Liu, Haipeng (State Key Laboratory of Marine Environmental Science, College of Oceanography and Environmental Science, Xiamen University) ;
  • Ma, Xingyuan (School of Biotechnology and State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology)
  • Received : 2017.04.06
  • Accepted : 2017.05.18
  • Published : 2017.08.28
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Abstract

Escherichia coli heat-labile enterotoxin (LT) and its non-toxic mutant (LTm) are well-known powerful mucosal adjuvants and immunogens. However, the yields of these adjuvants from genetically engineered strains remain at extremely low levels, thereby hindering their extensive application in fundamental and clinical research. Therefore, efficient production of these adjuvant proteins from genetically engineered microbes is a huge challenge in the field of molecular biology. In order to explore the expression bottlenecks of LTm in E. coli, we constructed a series of recombinant plasmids based on various considerations and gene expression strategies. After comparing the protein expression among strains containing different recombinant plasmids, the signal sequence was found to be critical for the expression of LTm and its subunits. When the signal sequence was present, the strong hydrophobicity and instability of this amino acid sequence greatly restricted the generation of subunits. However, when the signal sequence was removed, abundantly expressed subunits formed inactive inclusion bodies that could not be assembled into the hexameric native form, although the inclusion body subunits could be refolded and the biological activity recovered in vitro. Therefore, the dilemma choice of signal sequence formed bottlenecks in the expression of LTm. These results reveal the expression bottlenecks of LTm, provide guidance for the preparation of LTm and its subunits, and certainly help to promote efficient preparation of this mucosal adjuvant protein.

Keywords

References

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