Biotechnology and Bioprocess Engineering 2019; 24(6): 934-941  
Coexpression of Kex2 Endoproteinase and Hac1 Transcription Factor to Improve the Secretory Expression of Bovine Lactoferrin in Pichia pastoris
Jie Sun1, Jie Jiang1, Xinyang Zhai2, Shaoming Zhu2, Zhenzhen Qu1, Wei Yuan1, Zhao Wang1, and Chun Wei1,*
1Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China
2Changhai Biological Company, Zhejiang Medicine Co., Ltd., Shaoxing 312000, China
Correspondence to: Chun Wei*
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China
Tel: +86-571-88320781
E-mail: springweispring@163.com
Received: May 11, 2019; Revised: July 22, 2019; Accepted: August 11, 2019; Published online: December 31, 2019.
© The Korean Society for Biotechnology and Bioengineering. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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 Hac1p and α-signal peptide-cutting protease Kex2p with different promoters. Results showed that the inducible coexpression of Hac1p 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 Hac1 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, Hac1p, secretory expression, Pichia pastoris


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