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


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.


bovine lactoferrin Kex2p Haclp, secretory expression Pichia pastoris 


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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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