Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
권호 표지

Expression and Antibacterial Activity of Recombinant Human Lactoferrin in Methylotrophic Yeast, Pichia pastoris

Methylotrophic Yeast, Pichia pastoris에서 사람 락토페린의 발현 및 항균성 연구

  • Lee Sang O (Department of Bioscience and Biotechnology, College of Natural Sciences, Hankook University of Foreign Studies) ;
  • Im Eun Mi (Department of Bioscience and Biotechnology, College of Natural Sciences, Hankook University of Foreign Studies) ;
  • Nam Eun Joo (Department of Bioscience and Biotechnology, College of Natural Sciences, Hankook University of Foreign Studies) ;
  • Lee Hyune Hwan (Department of Bioscience and Biotechnology, College of Natural Sciences, Hankook University of Foreign Studies)
  • 이상오 (한국외국어대학교 자연과학대학 생명공학과) ;
  • 임은미 (한국외국어대학교 자연과학대학 생명공학과) ;
  • 남은주 (한국외국어대학교 자연과학대학 생명공학과) ;
  • 이현환 (한국외국어대학교 자연과학대학 생명공학과)
  • Published : 2004.12.01
Download PDF
⟨ Previous Next ⟩

Abstract

The expression and antibacterial. activity of recombinant human lactoferrin (hLf) was studied from meth­ylotrophic yeast Pichia pastoris. The gene encoding hLf, isolated from human breast cDNA library, was subcloned into the expression vector, pPIC3.5K under the control of AOX1 promoter. The gene was integrated into the host chromosome and was identified by Southern blotting. The expression of the integrated gene was investigated by RT-PCR, Northern blotting, SDS-PAGE and Western blotting. Discrete band corresponding to hLf was detected from the SDS-PAGE, which was confirmed by Western blotting. The expression was also confirmed by RT-PCR and Northern blotting. The antibacterial activity of the recombinant hLf (rhLf) was investigated using Staphy­lococcus aureus ATCC 6538P and Micrococcus flavus ATCC 10240 as test organisms. The rhLf showed strong antibacterial activities against the bacteria. Furthermore, many Gram-negative animal pathogens such as E.coli ATCC8739, 25922, and Salmonella typhimurium 114 and 115, Pseudomonas fluorescens ID 963 I, P. aeruginosa KCCM 11802, and Gram-positive bacteria Bacillus mesentericus were also inhibited in their growth by the rhLf.

사람의 모유에 많이 함유된 human lactoferrin(hLF)은 항균 및 항 바이러스 작용이 있는 것으로 보고되고 있다. 본 연구에서는 hLf를 메탄올자화 효모인 Pichia pastoris에 cloning하고 그 발현을 RT-PCR, Northern blotting, SDS-PAGE및 Western blotting으로 확인하였다. 그 결과 2.1 kb의 hLf 유전자가 P.pastoris의 염색체 DNA로 끼어들어가 안정적으로 hLf를 발현하였다. 이 재조합 P.pastotis로부터 hLf를 포함하는 세포 추출액을 얻어 항균 작용을 연구하였다. 발현된 재조합 hLf는 Staphylococcus aureus, Micrococcus flavus 등의 그람 양성균에 대해 강력한 항균작용을 보일 뿐만 아니라 그람 음성 동물성 병원균인 Pseudomonas fluorescens ID 9631, E. coli ATCC8739, 25922,35 등과 Salmonella typhimurium 114,115 등 다양한 균에 대해서도 강력한 항균작용을 보였다. 이는 재조합 hLf가 생물학적 활성이 있다는 것을 보여준다.

Keywords

References

  1. Ainscough, E.W., A.M. Brodie, and J.E. Plowman. 1979. The chromium, manganese, cobalt and copper complexes of human lactoferrin. Inorg. Chim. Acta. 33, 149-153
  2. Aisen, P. and I. Listowsky. 1980. Iron transport and storage proteins. Annu. Rev. Biochem. 49, 357-393
  3. Akin, D.T., M.Q. Lu, S. Kendall, J. Rundegren, and R.R. Arnold. 1994. Bactericidal activity of different forms of lactoferrin. Adv. Exp. Med. Biol. 357, 61-70
  4. Anderson, B.F., H. Baker, M. Norris, G.E. Rice, and D.W. Bake. 1989. Structure of human lactoferrin; Crystallographic structure analysis and refinement at 2.8Å resolution. J. Mol. Biol. 209, 711-734
  5. Arnold, R.R., M. Brewer, and J.J. Gauthier. 1980. Bactericidal activity of human lactoferrin : sensitivity of a variety of microorganisms. Infect. Immun. 28, 893-898
  6. Arnold, R.R., J.E. Russell, W.J. Champion, M. Brewer, and J.J. Gauthier. 1982. Bactericidal activity of human lactoferrin: Differentiation from the stasis of iron deprivation. Infect. Immun. 35, 792-799
  7. Cregg, J.M., J.L. Cereghino, J. Shi, and D.R. Higgings. 2000. Recombinant protein expression in Pichia pastoris. Mol. Biotechnol. 16, 23-52
  8. Doring, G., T. Pfestrof, K. Botzenhart, and M.A. Abdallah. 1988. Iron-chelating substances and inflammation. Scand. J. Gastroenterol. 23, 68-69
  9. Ellison III, R.T. and T.J. Giehl. 1991. Killing of gram-negative bacteria by lactoferrin and lysozyme. J. Clin. Invest. 88, 1080 - 1091
  10. Ellison III, R.T., T.J. Giehl, and F.M. LaForce. 1988. Damage of the outer membrane of the enteric Gram-negative bacteria by lactoferrin and transferrin. Infect. Immun. 56, 2774-2781
  11. Ikeda, M., A. Nozaki, K. Sugiyama, T. Tanaka, A. Naganuma, K. Tanaka, H. Sekihara, K. Shimotohno, M. Saito, and N. Kato. 2000. Characterization of antiviral activity of lactoferrin against hepatitis C virus infection in human cultures cells. Vir. Res. 66, 51-63
  12. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685
  13. Moriuchi, M. and H. Moriuchi. 2001. A milk protein lactoferrin enhances Human T Cell Leukemia Virus Type 1 and suppresses HIV-1 infection. J. Immunol. 166, 4231-4236
  14. Romanos, M.A., C.A. Scorer, and J.J. Clare. 1992. Foreign gene expression in yeast: review. Yeast 8, 423-488
  15. Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual 1st ed. Cold Spring Harbor Laboratory, New York
  16. Schmitt, M.E., A.B. Timothy, and B.L. Trumpower. 1990. A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic. Acids Res. 18, 3091-3092
  17. Schorer, C.A., J.J. Clare, W.R. McCombie, M.A. Romanos, and K. Sreekrishna. 1994. Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression. Biotechnology 12, 181-184
  18. Sreekrishna, K., J.F. Tschopp, and M. Fuke. 1987. Invertase gene (SUC2) of Saccharomyces cerevisiae as a dominant marker for transformation Pichia pastoris. Gene 59, 115-125
  19. Tschopp, J.F., G. Dverlow, R. Kosson, W. Craig, and L. Grinna. 1987. High-level secretion of glycosylated invertase in the methylotrophic yeast, Pichia pastoris. Biotechnology 5, 1305-1308