Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Expression of Human Heavy-Chain and Light-Chain Ferritins in Saccharomyces cerevisiae for Functional Foods and Feeds

Saccharomyces cerevisiae을 이용한 사람의 H-, L-ferritins 발현 연구

  • Han, Hye-Song (Institute of Life Sciences & Resources and Department of Food Science and Biotechnology, Kyung Hee University) ;
  • Lee, Joong-Lim (Institute of Life Sciences & Resources and Department of Food Science and Biotechnology, Kyung Hee University) ;
  • Park, Si-Hong (Institute of Life Sciences & Resources and Department of Food Science and Biotechnology, Kyung Hee University) ;
  • Kim, Jae-Hwan (Institute of Life Sciences & Resources and Department of Food Science and Biotechnology, Kyung Hee University) ;
  • Kim, Hae-Yeong (Institute of Life Sciences & Resources and Department of Food Science and Biotechnology, Kyung Hee University)
  • 한혜송 (경희대학교 생명자원과학연구원, 생명과학대학 식품생명공학과) ;
  • 이중림 (경희대학교 생명자원과학연구원, 생명과학대학 식품생명공학과) ;
  • 박시홍 (경희대학교 생명자원과학연구원, 생명과학대학 식품생명공학과) ;
  • 김재환 (경희대학교 생명자원과학연구원, 생명과학대학 식품생명공학과) ;
  • 김해영 (경희대학교 생명자원과학연구원, 생명과학대학 식품생명공학과)
  • Published : 2008.09.28
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Abstract

To produce human ferritins in yeast, human H-chain and L-chain ferritins were amplified from previously cloned vectors. Each amplified ferritin gene was inserted into the pYES2.1/V5-His-TOPO yeast expression vector under the control of the GAL1promoter. Western blot analysis of the recombinant yeast cells revealed that H-and L-chain subunits of human ferritin were expressed in Saccharomyces cerevisiae. Atomic absorption spectrometry (AAS) analysis demonstrated that the intracellular content of iron in the ferritin transformant was 1.6 to 1.8-fold higher than that of the control strain. Ferritin transformants could potentially supply iron-fortified nutrients for food and feed.

효모에서 사람의 H-, L-ferritin을 생산하기 위해서, 기존에 복제된 vector를 사용하였으며,단백질을 발현하기 위해서 각각의 증폭된 ferritin 유전자를 GALI promoter에 의해 조절되는 pYES2.1/V5-His-TOPO 효모 발현 vector에 삽입하였다. Western blot 분석을 통해서 사람의 H-, L-ferritin subunits을 함유한 재조합 효모에서 사람의 ferritin이 발현된 것을 확인할 수 있었다. 또한 Atomic absorption spectrometry(AAS) 분석을 통해서 형질변환된 효모의 철 함유량이 대조군과 비교하여 $1.6{\sim}l.8$배 증가한 것을 확인하였다. 향후 ferritin이 함유된 형질변환 효모를 사용하여 잠재적으로 철이 강화된 영양성분을 기능성 식품과 사료에 이용할 수 있을 것이다.

Keywords

References

  1. Beaumont, C., S. V. Torti, F. M. Torti, and W. H. Massover. 1996. Novel properties of L type polypeptide subunits in mouse ferritin molecules. J. Bio Chem. 271: 7923-7926 https://doi.org/10.1074/jbc.271.14.7923
  2. Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  3. Ford, G. C., P. M. Harrison, D. W. Rice, J. M. Smith, A. Treffry, J. L. White, and J. Yariv. 1984. Ferritin: Design and formation of an iron-storage molecule. Philos Trans R Soc Lond B Bio Sci. 304: 551-565 https://doi.org/10.1098/rstb.1984.0046
  4. Guo, J. H., M. Abedi, and S. D. Aust. 1996. Expression and loading of recombinant heavy and light chain homopolymers of rat liver ferritin. Arch. Biochem. Biophys. 335: 197-204 https://doi.org/10.1006/abbi.1996.0498
  5. Harrison, P. M. and P. Arosio. 1996. The ferritins: Molecular properties, iron storage function and cellular regulation. Biochim Biophys Acta 1275: 161-203 https://doi.org/10.1016/0005-2728(96)00022-9
  6. Kim, H. J., H. M. Kim, J. H. Kim, K. S. Ryu, S. M. Park, K. Y. Jahng, M. S. Yang, and D. H. Kim. 2003. Expression of heteropolymeric ferritin improves iron storage in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 69: 1999-2005 https://doi.org/10.1128/AEM.69.4.1999-2005.2003
  7. Lee, J. L., H. S. Song, H. J. Kim, D. K. Chung, C. S. Park, D. I. Jeoung, and H. Y. Kim. 2003. Functional expression and production of human H-ferritin in Pichia pastoris. Biotech. Lett. 25: 1019-1023 https://doi.org/10.1023/A:1024193104858
  8. Lee, J. L., S. N. Yang, C. S. Park, D. I. Jeoung, and H. Y. Kim. 2004. Purification and its glycosylation pattern of recombinant L-ferritin in Pichia pastoris. J. Microbio. Biotechnol. 14: 68-73
  9. Lee, J. L., C. S. Park, and H. Y. Kim. 2007. Functional assembly of recombinant human ferritin subunits in Pichia pastoris. J. Microbiol. Biotechnol. 17: 1695-1699
  10. Lee, J. L., and H. Y. Kim. 2008. Expression and iron storage properties of recombinant human ferritins in Escherichia coli: relevance for functional ferritins in food systems. Food Biotechnol. 22: 140-149 https://doi.org/10.1080/08905430802043131
  11. Levi, S., A. Luzzago, G. Cesareni, A. Cozzi, F. Franceschinelli, A. Albertini, and P. Arosio. 1988. Mechanism of ferritin iron uptake: Activity of the H-chain and deletion mapping of the ferro-oxidase site. A study of iron uptake and ferro-oxidase activity of human liver, recombinant H-chain ferritins, and of two H-chain deletion mutants. J. Biol. Chem. 263: 18086-18092
  12. Levi, S., J. Salfeld, F. Franceschinelli, A. Cozzi, M. H. Dorner, and P. Arosio. 1989. Expression and structural and functional properties of human ferritin L-chain from Escherichia coli. Biochemistry. 28: 5179-5184 https://doi.org/10.1021/bi00438a040
  13. Levi, S., P. Santambrogio, A. Cozzi, E. Rovida, B. Corsi, E. Tamborini, S. Spada, A. Albertini, and P. Arosio. 1994. The role of the L-chain in ferritin iron incorporation. Studies of homo and heteropolymers. J. Mol. Biol. 238: 649-654 https://doi.org/10.1006/jmbi.1994.1325
  14. Levi, S., P. Santambrogio, B. Corsi, A. Cozzi, and P. Arosio. 1996. Evidence that residues exposed on the three-fold channels have active roles in the mechanism of ferritin iron incorporation. Biochem J. 317: 467-473 https://doi.org/10.1042/bj3170467
  15. Munro, H. 1993. The ferritin genes: their response to iron status. Nutr. Rev. 51: 65-73 https://doi.org/10.1111/j.1753-4887.1993.tb03072.x
  16. Orino, K., K. Eguchi, T. Nakayama, S. Yamamoto, and K. Watanabe. 1997. Sequencing of cDNA clones that encode bovine ferritin H and L chains. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 118: 667-673 https://doi.org/10.1016/S0305-0491(97)00277-0
  17. Rucker, P., F. M. Torti, and S. V. Torti. 1996. Role of H and L subunits in mouse ferritin. J. Biol. Chem. 271: 33352-33357 https://doi.org/10.1074/jbc.271.52.33352
  18. Santambrogio, P., A. Cozzi, S. Levi, E. Rovida, F. Magni, A. Albertini, and P. Arosio. 2000. Functional and immunological analysis of recombinant mouse H- and L-ferritins from Escherichia coli. Protein Expr. Purif. 19: 212-218 https://doi.org/10.1006/prep.2000.1212
  19. Shin, Y. M., T. H. Kwon, K. S. Kim, K. S. Chae, D. H. Kim, J. H. Kim, and M. S. Yang. 2001. Enhanced iron uptake of Saccharomyces cerevisiae by heterologous expression of a tadpole ferritin gene. Appl. Environ. Microbiol. 67: 1280-1283 https://doi.org/10.1128/AEM.67.3.1280-1283.2001
  20. Takeda, S., M. Yamaki, S. Ebina, and K. Nagayama. 1995. Site-specific reactivities of cysteine residues in horse L-apoferritin. J. Biochem. (Tokyo). 117: 267-270 https://doi.org/10.1093/jb/117.2.267
  21. Wade, V. J., S. Levi, P. Arosio, A. Treffry, P. M. Harrison, and S. Mann. 1991. Influence of site-directed modifications on the formation of iron cores in ferritin. J. Mol. Biol. 221: 1443-1452 https://doi.org/10.1016/0022-2836(91)90944-2
  22. Waldo, G. S., and E. C. Theil. 1993. Formation of iron(III)- tyrosinate is the fastest reaction observed in ferritin. Biochemistry. 32: 13262-13269 https://doi.org/10.1021/bi00211a039
  23. Worwood, M. 1990. Ferritin. Blood Rev. 4: 259-269 https://doi.org/10.1016/0268-960X(90)90006-E