Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research Paper
  • Published:

Production of Biologically Active Recombinant Human Lactoferrin in Aspergillus Oryzae

Bio/Technology volume 10, pages 784–789 (1992)Cite this article

Abstract

We report the production of recombinant human lactoferrin in Aspergillus oryzae. Expression of human lactoferrin (hLF), a 78 kD glycoprotein, was achieved by placing the cDNA under the control of the A. oryzae α–amylase promoter and the 3′ flanking region of the A. niger glucoamylase gene. Using this system, hLF is expressed and secreted into the growth medium at levels up to 25 mg/l. The recombinant lactoferrin is indistinguishable from human milk lactoferrin with respect to its size, immunoreactivity, and iron–binding capacity. The recombinant protein appears to be appropriately N–linked glycosylated and correctly processed at the N–terminus by the A. oryzae secretory apparatus. Lactoferrin is the largest heterologous protein and the first mammalian glycoprotein expressed in the Aspergillus system to date. Hence, this expression system appears suitable for the large–scale production and secretion of biologically active mammalian glycoproteins.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. William, J. 1985. The structure of transferrins, p. 13–23. In: Proteins of Iron Storage and Transport. Spik. G. Montreuil, J., Crichton. R. R. and Mazurier, J. (Eds.). Eisevier. Amsterdam.

    Google Scholar 

  2. Groves, M.L. 1960. The isolation of a red protein from milk. J. Am. Chem. Soc. 82: 3345–3350.

    Article  CAS  Google Scholar 

  3. Masson, P.L., Heremans, J.F. and Dive, C. 1966. An iron-binding protein common to many external secretions. Clin. Chim. Acta. 14: 735–739.

    Article  CAS  Google Scholar 

  4. Masson, P.L., Heremans, J.F. and Schonne, E. 1969. Lactoferrin. an iron-binding protein in neutrophilic leukocytes. J. Exp. Med. 130: 643–658.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Briggs, R.C., Glass, I.J., Montiel, W.F., M.M. and Hinclica, L.S. 1981. Lactoferrin: Nuclear localization in the human neutrophilic granulotyte. J. Histochem. Cytochem. 29: 1182–1186.

    Article  Google Scholar 

  6. Metz-Boutigue, M.H., Jolles, J., Mazurier, J., Schoentgen, K., Legrand, D., Spik, G., Montreuil, J. and Jolles, P. 1984. Human lai loferrin: amino acid sequence and structural comparisons with other transferrins. Eur. J. Biochem. 145: 659–676.

    Article  CAS  PubMed  Google Scholar 

  7. Anderson, B.F., Baker, H.M., Norris, G.F., Rice, D.W. and Baker, E.N. 1989. Structure of human lactoferrin: Crystallographic structure-analysis and refinement at 2.8 A resolution. J. Mol. Biol. 209: 711–734.

    Article  CAS  PubMed  Google Scholar 

  8. Schlabach, M.R. and Bates, G.W. 1975. The synergistic binding of anions and Fe3+ by transferring. J. Biol. Chem. 250: 2182–2188.

    CAS  PubMed  Google Scholar 

  9. Anderson, B.F., Anderson, B.F., Baker, H.M., Norris, G.E., Rumball,and Baker, F.N. 1990. Apolactoferrin structure demonstrates ligand-induced conformational change in transferrins. Nature 344: 784–787.

    Article  CAS  PubMed  Google Scholar 

  10. Arnold, R.R., Cole, M.F. and McGhee, J.R. 1977. A bacteriocidal effect for human lactoferrin. Science 197: 263–265.

    Article  CAS  PubMed  Google Scholar 

  11. Nemet, K. and Simonovits, I. 1985. The biological role of lactoferrin. Haematol. 18: 3–12.

    CAS  Google Scholar 

  12. Hashizume, S., Kuroda, K. and Murakami, M. 1983. Identification of lactoferrin as an essential growth factor for human lymphatic cells lines in serum free medium. Biochim. Biophys. Acta 763: 377–382.

    Article  CAS  PubMed  Google Scholar 

  13. Broxemeyer, H.E., De Sousa, M., Smithyman, A., Ralph, P., Hamilton, J., Kurland, J. and Bognacki, 1980. Specificity and modulation of the action of lactoferrin, a negative feedback regulator of myelopoiesis. Blood 55: 324–333.

    Google Scholar 

  14. Oseas, R., Yang, H.H., Baehner, R.L. and Boxer, L.A. 1981. Lactoferrin: A promoter of polymorphonuclear leukocyte adhesiveness. Blood 57: 939–945.

    CAS  PubMed  Google Scholar 

  15. Reiter, B. 1978. Review of nonspecific and microbial factors in milk. Ann. Rech. Vet. 9: 205–224.

    CAS  PubMed  Google Scholar 

  16. Barbesgaard, P. 1977. Industrial enzymes produced by members of the genus Aspergillus, p. xxx–xxx? In: Genetics and Physiology of Aspergillus. Smith, J. E. and Pateman, J. A. (Eds.). Academic Press, London.

    Google Scholar 

  17. Van Brunt, J. 1986. Fungi, the perfect hosts? Bio/Technology 4: 1057–1062.

    Article  CAS  Google Scholar 

  18. Sanders, G., Picknett, T.M., Tuite, M.F. and Ward, M. 1989. Heterologous gene expression in filamentous fungi. Trends in Biotechnol. 7: 283–287.

    Article  Google Scholar 

  19. Christensen, T., Woeldike, H., Boel, E., Mortensen, S.B., Hjortschoej, K., Thim, L. and Hansen, M.T. 1988. High level expression of recombinant genes in Aspergillus oryzae . Bio/Technology 6: 1419–1422.

    CAS  Google Scholar 

  20. Gines, M.J., Dove, M.J. and Seligy, U.L. 1989. Aspergillus oryzae has two nearly identical TAKA-amylase genes, each containing eight introns. Gene 79: 107–117.

    Article  CAS  PubMed  Google Scholar 

  21. Boel, E., Hansen, M.T., Hjort, I., Hoegh, I. and Fiil, N.P. 1984. Two different types of intervening sequences in the glucoamylase gene from Aspergillus niger . EMBO J. 3: 1581–1585.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Waring, R.B., May, G.S. and Morris, R.N. 1989. Characterization of an inducible expression system in Aspergillus nidulans using alcA and tubulin-coding genes. Gene 79: 119–130.

    Article  CAS  PubMed  Google Scholar 

  23. Erratt, J.A., Douglas, P.E., Moranelli, F. and Seligy, U.L. 1984. The induction of α-amylase by starch in Aspergillus oryzae: evidence for controlled mRNA expression. Can. J. Biochem. Cell. Biol. 62: 678–690.

    Article  CAS  PubMed  Google Scholar 

  24. Huge-Jensen, B., Andreasen, F., Christensen, T., Christensen, M., Thim, L. and Boel, E. 1989. Rhizomucor miehei triglyceride lipase is processed and secreted from transformed Aspergillus oryzae . Lipids 24: 781–785.

    Article  CAS  PubMed  Google Scholar 

  25. Vilja, P., Krohn, K. and Tuohimaa, P. 1985. A rapid and sensitive non-competitive avidin-biotin assay for lactoferrin. J. Immunol. Methods. 76: 73–83.

    Article  CAS  PubMed  Google Scholar 

  26. Stowell, K.M., Rado, T.A., Funk, W.D. and Tweedie, J.W. 1991. Expression of cloned human lactoferrin in baby-hamster kidney cells. Biochem. J. 276: 349–355.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Hutchens, W.T., Henry, J.F. and Yip, T.-T. 1991. Structurally intact (78-kDa) forms of maternal lactoferrin purified from urine of preterm infants fed human milk: Identification of a trypsin-like proteolytic cleavage event in vivo that does not result in fragment dissociation. Proc. Natl. Acad. Sci. U.S.A. 88: 2994–2998.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Spik, G., Coddenville, B. and Montreuil, J. 1988. Comparative study of the primary structures of sero, lacto- and ovatransferrin glycans from different species. Biochimie. 70: 1459–1469.

    Article  CAS  PubMed  Google Scholar 

  29. Tarentino, A.L., Gornez, C.M. and Plummer, T.H. 1985 Deglycosylation of asparagine-linked glycans by peptide: N-glycosidase F. Biochemistry 24: 4665–4671.

    Article  CAS  PubMed  Google Scholar 

  30. Ward, M., Wilson, L.J., Kodama, K.H., Rey, M.W. and Berka, R.M. 1990. Improved production of chymosin in Aspergillus by expression as a glucoamylase-chymosin fusion. Bio/Technology 8: 435–439.

    CAS  Google Scholar 

  31. Dunn-Coleman, N.S., Bloebaum, P., Berka, R.M., Bodie, E., Robinson, N., Armstrong, G., Ward, M., Przetak, M., Carter, G.L., LaCost, R., Wilson, L.J., Kodama, K.H., Baliu, E.F., Bower, B., Lamsa, M. and Heinsohn, H. 1991. Commercial levels of chymosin production by Aspergillus . Bio/Technology 9: 976–981.

    Article  CAS  Google Scholar 

  32. Krimperfort, P., Rademakers, A., Eyestone, W., van der Schans, A., van der Broek, S., Kootwijk, E., Platenburg, G., Pieper, F., Strijker, R. and de Boer, H. 1991. Generation of transgenic dairy cattle using in vitro embryo production. Bio/Technology 9: 844–847.

    Google Scholar 

  33. Bal, J., Kajtaniak, E.M. and Pieniazek, N.J. 1977. 4-Nitroquinoline-l-oxide: A good mutagen for Aspergillus nidulans . Mutation Res. 56: 153–156.

    Article  CAS  Google Scholar 

  34. Osmani, S.A., May, G.S. and Morris, N.R. 1987. Regulation of the mRNA levels of nimA, a gene required for the G2-M transition in Aspergillus mdulans . J. Cell. Biol. 104: 1495–1504.

    Article  CAS  PubMed  Google Scholar 

  35. Rado, T.A., Wei, X. and Benz, E.J. 1987. Isolation of lactoferrin cDNA from a human myeloid library and expression of mRNA during normal and leukemic myelopoiesis. Blood 70: 989–993.

    CAS  PubMed  Google Scholar 

  36. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487–491.

    Article  CAS  PubMed  Google Scholar 

  37. Rasmussen, C.D., Means, R.L., Lo, K.P., May, G.S. and Means, A.R. Characterization and expression of the unique calmodulin gene of Aspergillus nidulans . J. Biol. Chem. 265: 13767–13775.

  38. Maniatis, T., Fritsh, E.F. and Sambrook, J. 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

  39. Thomas, P. 1980. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 77: 5201–5205.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Author notes

  1. Orla M. Conneely: Corresponding author.

Authors and Affiliations

  1. Department of Cell Biology, Baylor College of Medicine, Houston, Texas, 77030

    Pauline P. Ward, Jing-Y. Lo, Gregory S. May & Orla M. Conneely

  2. Cell and Molecular Biology Group, Department of Biochemistry, University College, Galway, Ireland

    Mairead Duke & Denis R. Headon

Authors
  1. Pauline P. Ward
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jing-Y. Lo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mairead Duke
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gregory S. May
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Denis R. Headon
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Orla M. Conneely
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ward, P., Lo, JY., Duke, M. et al. Production of Biologically Active Recombinant Human Lactoferrin in Aspergillus Oryzae. Nat Biotechnol 10, 784–789 (1992). https://doi.org/10.1038/nbt0792-784

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt0792-784

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing