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Application of immobilized thrombin for production of S-thanatin expressed in Escherichia coli

Abstract

S-thanatin, a small antimicrobial peptide with 21 amino acid residues, was expressed as a fusion protein containing thrombin cleavage site in Escherichia coli BL21 (DE3). To reduce the production cost, immobilization of thrombin in polyacrylamide gel for cleavage was studied in this work. The immobilized thrombin exhibited excellent activity within wider ranges of pH value and temperature for reaction than free enzyme, and the residual activity could remain above 75% after ten times of usage. Tricine–SDS–PAGE result showed that the immobilized thrombin could cleave the S-thanatin fusion protein effectively. After cleavage, recombinant S-thanatin was purified by preparative reversed-phase high-performance liquid chromatography and mass spectrum showed that the molecular weight (2,448.86) was close to the theoretical value (2,448.98). After purification, about 7 mg of S-thanatin was obtained from 1 l of culture and the recombinant exhibited excellent bioactivity to E. coli ATCC 25922, with the minimum inhibitory concentration of 12 μg/ml. The purification method could be applied to prepare other peptides with similar properties at low cost.

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Acknowledgments

We thank the Natural Science Foundation of Jiangsu Province, China (No. BK2009274) and the Science Foundation of Southeast University (No. 3290000102) for the financial support for this work. Meanwhile, Dr. Xiaobo Fan and Dr. Jing Xiong are thanked for the careful revision of this paper.

Author information

Correspondence to Guoqiu Wu or Hanmei Xu.

Additional information

Guoqiu Wu and Xuepeng Deng contributed equally to this article.

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Wu, G., Deng, X., Li, X. et al. Application of immobilized thrombin for production of S-thanatin expressed in Escherichia coli . Appl Microbiol Biotechnol 92, 85–93 (2011). https://doi.org/10.1007/s00253-011-3379-z

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Keywords

  • Antimicrobial peptide
  • Thanatin
  • Immobilized thrombin
  • Fusion expression
  • Escherichia coli