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Coexpression of Kex2 Endoproteinase and Hac1 Transcription Factor to Improve the Secretory Expression of Bovine Lactoferrin in Pichia pastoris

  • Jie Sun
  • Jie Jiang
  • Xinyang Zhai
  • Shaoming Zhu
  • Zhenzhen Qu
  • Wei Yuan
  • Zhao Wang
  • Chun WeiEmail author
Research Paper
  • 26 Downloads

Abstract

The large-scale production of functional recombinant lactoferrin has become a major goal because of its medicinal value and global demand. Secreting recombinant proteins into a culture medium offers a way to simplify protein purification and avoid toxicity from intracellularly accumulated materials. In this study, after 84 h of induction with methanol in a shaking flask, the recombinant bovine lactoferrin (rbLf) titer in the culture supernatant of the strain that integrated two copies of the rbLf gene was only 121.6 μg/L. A bottleneck might have existed in the folding and secretion pathways of rbLf. We then attempted to further improve the rbLf titer by overexpressing the transcription factor Haclp and α-signal peptide-cutting protease Kex2p with different promoters. Results showed that the inducible coexpression of Haclp and Kex2p linked with the 2A sequence improved the rbLf titer 5.0-fold (735.8 μg/L) after 84 h of induction with methanol. The maximal titer in a shaking flask was 1,150.5 μg/L after 120 h of induction. The rbLf titer achieved 35.6 mg/L in a 5 L fed-batch fermenter. Thus, Kex2 and Hacl overexpression driven by methanol-induced promoter alleviated the bottleneck in the folding and secretion pathways and greatly improved the secretory expression of rbLf in Pichia pastoris.

Keywords

bovine lactoferrin Kex2p Haclp, secretory expression Pichia pastoris 

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Notes

Acknowledgements

This work was financially supported by the Zhejiang Provincial Natural Science Foundation of China (Grant Nos. LY19C010005 and LQ18C010006).

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Copyright information

© The Korean Society for Biotechnology and Bioengineering and Springer 2019

Authors and Affiliations

  • Jie Sun
    • 1
  • Jie Jiang
    • 1
  • Xinyang Zhai
    • 2
  • Shaoming Zhu
    • 2
  • Zhenzhen Qu
    • 1
  • Wei Yuan
    • 1
  • Zhao Wang
    • 1
  • Chun Wei
    • 1
    Email author
  1. 1.Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and BioengineeringZhejiang University of TechnologyHangzhouChina
  2. 2.Changhai Biological CompanyZhejiang Medicine Co., Ltd.ShaoxingChina

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