Applied Biochemistry and Biotechnology

, Volume 179, Issue 1, pp 33–45 | Cite as

A Novel Method for Efficient Preparation of Mucosal Adjuvant Escherichia coli Heat-Labile Enterotoxin Mutant (LTm) by Artificially Assisted Self-Assembly In Vitro

  • Di Liu
  • Na Zhang
  • Wenyun Zheng
  • Hua Guo
  • Xiaoli Wang
  • Tianwen Wang
  • Ping WangEmail author
  • Xingyuan MaEmail author


As well-known powerful mucosal adjuvant proteins, Escherichia coli heat-labile enterotoxin (LT) and its non-toxic or low-toxic mutants (LTm) are capable of promoting strong mucosal immune responses to co-administered antigens in various types of vaccines. However, due to the complex composition and special structure, the yield of LTm directly from the recombinant genetic engineering strains is quite low. Here, we put forward a novel method to prepare LTm protein which designed, expressed, and purified three kinds of component subunits respectively and assembled them into a hexamer structure in vitro by two combination modes. In addition, by simulated in vivo environment of polymer protein assembly, the factors of the protein solution system which include environment temperature, pH, ionic strength of the solution, and ratio between each subunit were taken into consideration. Finally, we confirmed the optimal conditions of two assembly strategies and prepared the hexamer holotoxin in vitro. These results are not only an important significance in promoting large-scale preparation of the mucosal adjuvant LTm but also an enlightening to produce other multi-subunit proteins.


Heat-labile enterotoxin (LT) Mucosal adjuvant In vitro preparation Artificially assisted self-assembly 



This study was supported by the National Natural Science Foundation (30873190, 31300660), Science and Technology Innovation Action Plan of Shanghai (14431904300), Shanghai Pujiang Program (13PJD012), and a foundation for young teachers from Education Ministry of China (20120074120027) and partially supported by the Open Funding Project of the State Key Laboratory of Bioreactor Engineering.

Compliance of Ethical Standards

Conflict of Interest

The authors declare no conflicts of interest. The authors are responsible for the content and writing of the paper.


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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.School of Biotechnology and State Key Laboratory of Bioreactor engineeringEast China University of Science and TechnologyShanghaiChina
  2. 2.School of Pharmacy, Shanghai Key Laboratory of New Drug DesignEast China University of Science and TechnologyShanghaiChina

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