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Expression of human lactoferrin N-lobe in Pichia pastoris and its antibacterial activity

Pichia pastoris에서 사람 락토페린 N-lobe의 발현과 항균활성

  • Won, Su-Jin (Department of Bioscience and Biotechnology and Protein Research Center of GRRC, Hankuk University of Foreign Studies) ;
  • Jo, Jae-Hyung (Department of Bioscience and Biotechnology and Protein Research Center of GRRC, Hankuk University of Foreign Studies) ;
  • Kim, Seung-Hwan (Department of Bioscience and Biotechnology and Protein Research Center of GRRC, Hankuk University of Foreign Studies) ;
  • Kwon, Hyuk-Jin (Department of Bioscience and Biotechnology and Protein Research Center of GRRC, Hankuk University of Foreign Studies) ;
  • Lee, Hyune-Hwan (Department of Bioscience and Biotechnology and Protein Research Center of GRRC, Hankuk University of Foreign Studies)
  • 원수진 (한국외국어대학교 생명공학과) ;
  • 조재형 (한국외국어대학교 생명공학과) ;
  • 김승환 (한국외국어대학교 생명공학과) ;
  • 권혁진 (한국외국어대학교 생명공학과) ;
  • 이현환 (한국외국어대학교 생명공학과)
  • Received : 2015.09.02
  • Accepted : 2015.09.11
  • Published : 2015.09.30

Abstract

Lactoferrin (LF) is a multifunctional, iron-binding glycoprotein found in physiological secretions of mammals. LF shows antibacterial, antiviral and antifungal activities. In the present study, a gene encoding the N-terminal lobe of human lactoferrin (hLF) was isolated, cloned and expressed in methylotrophic yeast, Pichia pastoris. The recombinant hLF-N (rhLF-N) protein was secreted into the culture medium at the level of $458{\mu}g/ml$ in 3 L fermentor. The size of purified hLF-N was estimated as 35 kDa when analyzed by SDS-PAGE and western blotting. The rhLF-N was further confirmed by immunodiffusion using the anti-hLF polyclonal antibody. The expression profile analysis by qRT-PCR showed that the relative mRNA expression of rhLF-N was maximal after 2-3 days of methanol induction and reduced gradually at 4 days. The purified rhLF-N showed broad antibacterial activities against the pathogens such as Staphylococcus aureus, E. coli, Pseudomonas aeruginosa, Burkholderia cepacia, and Salmonella typhimurium. However, rhLF-N showed relatively lower activity when compared to peptides derived from LF. In spite of this weak activity, the rhLF-N expressed in P. pastoris might be more advantageous for the industrial application, because rhLF-N is secreted into the culture medium and the production can also be increased by optimization of culture conditions.

Keywords

Pichia pastoris;antibacterial activity;expression;human lactoferrin;N-lobe

Acknowledgement

Supported by : 한국외국어 대학교

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