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Efficient KEX2-Like Processing of a Glucoamylase-Interleukin-6 Fusion Protein by Aspergillus Nidulans and Secretion of Mature Interleukin-6

Bio/Technology volume 9, pages 378–381 (1991)Cite this article

Abstract

We have designed an expression vector for the secretion of human interleukin-6 (hIL-6) in which the mature protein is fused through a spacer peptide, contain-ing a KEX-2 like protein processing signal, to the entire Aspergillus niger glucoamylase (glaA) gene. Transformation of Aspergillus nidulans with this vector results in fungal strains secreting equimolar amounts of the glucoamylase and IL-6 proteins. The KEX2-type processing sig-nal, Lys-Arg, is recognized and cleaved efficiently by an enzyme present in A. nidulans resulting in the secretion of an authentic mature hIL-6 protein at levels of up to 5 mg/l.

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References

  1. Saunders, G., Picknett, T.M., Tuite, M.F. and Ward, M. 1989. Heterologous gene expression in filamentous fungi. Trends in Biotechnology 7: 283–287.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  3. Carrez, D., Janssens, W., Degrave, P., van den Hondel, C., Kinghorn, J., Fiers, W. and Contreras, R. 1991. Heterologous gene expression by filamentous fungi: secretion of human interleukin-6 by Aspergillus nidulans. Gene 94: 147–154.

    Article  Google Scholar 

  4. van den Hondel, C. et al. Unpublished results.

  5. Guisez, Y., Tison, B., Vandekerckhove, J., De Molder, J., Bauw, G., Fiers, W. and Contreras, R. 1991. Production and purification of recombinant human interleukin-6 secreted by the yeast Saccharomyces cerevisiae. Eur. J. Biochem. In press.

    Google Scholar 

  6. Finkelstein, D.B. 1987. Improvement of enzyme production in Aspergillus. Antonie van Leeuwenhoek J. Microbiol. 53: 349–352.

    Article  CAS  PubMed  Google Scholar 

  7. Gwynne, D.I., Buxton, F.P., Williams, S.A., Sillis, A.M. Johnstone, J.A., Buch, J.K., Guo, Z.-M., Drake, D., Westphal, M. and Davies, R.W. 1989. Development of an expression system in Aspergillus nidulans. Biochem. Soc. Trans. 17: 338–340.

    Article  CAS  PubMed  Google Scholar 

  8. Finkelstein, D.B., Rambosek, J.A., Leach, J., Wilson, R.E., Larson, A.E., McAda, P.C., Soliday, C.L. and Ball, C. 1986. Genetic transformation and protein secretion in industrial filamentous fungi. Fifth International Symposium on the genetics of industrial microorganisms. Alacevic, M., Hranueli, D., Toman, Z. (Eds.). Pliva Press, Zagreb.

    Google Scholar 

  9. Yelton, M.M., Hamer, J.E. and Timberlake, W.E. 1984. Transformation of Aspergillus nidulans by using a trpC plasmid. Proc. Natl. Sci. USA 81: 1470–1474.

    Article  CAS  Google Scholar 

  10. John, M.A. and Peberdy, J.F. 1984. Transformation of Aspergillus nidulans using the argB gene. Enzyme Microb. Technol. 6: 386–389.

    Article  CAS  Google Scholar 

  11. Dmochowska, A., Berse, B., Kowalska, I., Skrzypek, M. and Weglenski, P. 1986. Expression of the Aspergillus nidulans argB gene in Escherichia coli. J. Gen. Microbiol. 132: 1729–1737.

    CAS  PubMed  Google Scholar 

  12. Schaffner, W. and Weissmann, C. 1973. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal. Biochem. 56: 502–514.

    Article  CAS  PubMed  Google Scholar 

  13. Van Snick, J., Cayphas, S., Vink, A., Uyttenhove, C., Coulie, P.G., Rubira, M.R. and Simpson, R.J. 1986. Purification and NH2-terminal amino acid sequence of a T-cell-derived lymphokine with growth factor activity for B-cell hybridomas. Proc. Natl. Acad. Sci USA 83: 9679–9683.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Gross, V., Andus, T., Castell, J., Vom Berg, D., Heinrich, P.C. and Gerok, W. 1989. O- and N-glycosylation lead to different molecular mass forms of human monocyte interleukin-6. FEBS Lett. 247: 323–326.

    Article  CAS  PubMed  Google Scholar 

  15. Van Damme, J., Van Beeumen, J., Decock, B., Van Snick, J., De Ley, M. and Billiau, A. 1988. Separation and comparison of two mono-kines with lymphocyte-activating factor activity: IL-1β and hybridoma growth factor (HGF). J. Immunol. 140: 1534–1541.

    CAS  PubMed  Google Scholar 

  16. Svensson, B., Larsen, K., Svendsen, I. and Boel, E. 1983. The complete amino acid sequence of the glycoprotein, glucoamylase Gl, from Aspergillus niger. Carlsberg Res. Commun. 48: 529–544.

    Article  CAS  Google Scholar 

  17. Marston, F. 1986. The purification of eukaryotic polypeptides synthesized in E. coli. Biochem. J. 240: 1–12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Sabin, E., Lee-Ng, C.-T., Shuster, J. and Barr, P. 1989. High-level expression and in vivo processing of chimeric ubiquitin fusion proteins in Saccharomyces cerevisiae. Bio/Technology 7: 705–709.

    CAS  Google Scholar 

  19. Hinnen, A., Meyhack, B. and Tsapis, R. 1983. High expression and secretion of foreign proteins in yeast, p. 157–163. In: Gene Expression in Yeast. Korhula, M., Vaisanen, E. (Eds.). Proceedings of the Alko yeast symposium. Helsinki Foundation for Biotechnical and Industrial Fermentation Research, Helsinki, Finland.

    Google Scholar 

  20. Ducan, M., Kohno, T., Stashenko, K. and Yamasaki, E. 1984. Secretion of chymosin by S. cerevisiae using the invertase signal sequence. 12th Int. Conf. Yeast Genet. Mol. Biol. Abstract Q7, p. 296.

  21. Kingsman, S.M., Kingsman, A.J. and Mellor, J. 1987. The production of mammalian proteins in Saccharomyces cerevisiae. Trends Biotechnol. 553–56.

    Article  Google Scholar 

  22. Harkki, A., Uusitalo, J., Bailey, M., Penttilä, M. and Knowles, J.K.C. 1989. A novel fungal expression system: secretion of active calf chymosin from the filamentous fungus Trichoderma reesei. Bio/Technology 7: 596–603.

    CAS  Google Scholar 

  23. Ward, M., Wilson, L., Kodama, K., Rey, M. and Berka, R. 1990. Improved production of chymosin in Aspergillus by expression as a glucoamylase-chymosin fusion. Bio/Technology 8: 435–440.

    CAS  Google Scholar 

  24. Devchand, M., Gwynne, D., Buxton, F. and Davies, R. 1989. An efficient cell-free translation system from Aspergillus nidulans and in vitro translocation of prepro-α-mating factor across Aspergillus mi-crosomes. Curr. Genet. 14: 561–566.

    Article  Google Scholar 

  25. Sleep, D., Belfield, G. and Goodey, A. 1990. The secretion of human serum albumin from the yeast Saccharomyces cerevisiae using five different leader sequences. Bio/Technology 8: 42–46.

    CAS  Google Scholar 

  26. Innis, M.A., Holland, M.J., McCabe, P.C., Cole, G.F., Wittman, V.P., Tal, R., Watt, K.W.K., Gelfand, D.H., Holland, J.P. and Meade, J.H. 1985. Expression, glycosylation and secretion of an Aspergillus glucoamylase by Saccharomyces cerevisiae. Science 228: 21–26.

    Article  CAS  PubMed  Google Scholar 

  27. Thomas, G., Thorne, B., Thomas, L., Alien, R., Hruby, D., Fuller, R. and Thorner, J. 1988. Yeast KEX2 endopeptidase correctly cleaves a neuroendocrine prohormone in mammalian cells. Science 241: 226–231.

    Article  CAS  PubMed  Google Scholar 

  28. Kreil, G. 1990. Processing of precursors by dipeptidylaminopeptidases. Trends in Biochem. Sciences 15: 23–26.

    CAS  Google Scholar 

  29. Argos, P. 1990. An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion. J. Mol. Biol. 211: 943–958.

    Article  CAS  PubMed  Google Scholar 

  30. Casadaban, M.J., Chou, J. and Cohen, S.N. 1980. In vitro gene fusions that join an enzymatically active β-galactosidase segment to amino-terminal fragments of exogenous proteins: E. coli plasrnid vectors for the detection and cloning of translational initiation signals. J. Bacteriol. 143: 971–980.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Lynch, D. and Synder, S. 1986. Neuropeptides: multiple molecular forms, metabolic pathways, and receptors. Annu. Rev. Biochem. 55: 773–799.

    Article  CAS  PubMed  Google Scholar 

  32. Tyrer, M. 1986. Invertebrate neurobiology: catching up with peptides. Nature 322: 596–597.

    Article  Google Scholar 

  33. Fuller, R., Brake, A. and Thorner, J. 1989. Intracellular targeting and structural conservation of a prohormone-processing endoprotease. Science. 246: 482–486.

    Article  CAS  PubMed  Google Scholar 

  34. Bauw, G., De Loose, M., Inze, D., Van Montagu, M. and Vandekerckhove, J. 1987. Alterations in the phenotype of plant cells studied by NH2-terminal amino acid-sequence analysis of proteins electroblotted from two-dimensional gel-separated total extracts. Proc. Natl. Acad. Sci. USA. 84: 4806–4810.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Hopp, T.P. and Woods, K.R. 1981. Prediction of protein antigenic determinants from amino acid sequences. Proc. Natl. Acad. Sci. USA. 78: 3824–3828.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Pontecorvo, G., Roper, J.A., Hemmons, L.M., McDonald, K.D. and Bufton, A.W.J. 1953. The genetics of Aspergillus nidulans. Advances in Genetics 5: 141–238.

    Article  CAS  PubMed  Google Scholar 

  37. Hanahan, D. 1983. Studies on transformation of E. coli with plasmids. J. Mol. Biol. 166: 557–580.

    Article  CAS  PubMed  Google Scholar 

  38. Morinaga, Y., Franceschini, T., Inouye, S. and Inouye, M. 1984. Improvement of oligonucleotide-directed site-specific mutagenesis using double-stranded plasmid DNA. Bio/Technology 2: 636–639.

    CAS  Google Scholar 

  39. Zhu, J.D., Kempenaers, W. Van der Straeten, D, Contreras, R. and Fiers, W. 1985. A method for fast and pure DNA elution from agarose gels by centrifugal filtration. Bio/Technology 3: 1014–1016.

    CAS  Google Scholar 

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Author information

Author notes

  1. Dirk Carrez

    Present address: Solvay & Cie, Centraal Laboratorium, Dienst voor Biochemie LC207, Ransbeekstraat 310, B-1120, Brussel, Belgium

  2. Roland Contreras: Corresponding author.

Authors and Affiliations

  1. Laboratory of Molecular Biology, State University, Ledeganckstraat 35, B-9000, Gent, Belgium

    Roland Contreras & Walter Fiers

  2. University of St. Andrews, St. Andrews, Fife, KY16 9AL, Scotland, UK

    James R. Kinghorn

  3. Medisch Biologisch Laboratorium, TNO, P.O. Box 45, NL-2280 AA, Rijswijk, Nederland

    Cees A. M. J. J. van den Hondel

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  1. Roland Contreras
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  4. Cees A. M. J. J. van den Hondel
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  5. Walter Fiers
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Contreras, R., Carrez, D., Kinghorn, J. et al. Efficient KEX2-Like Processing of a Glucoamylase-Interleukin-6 Fusion Protein by Aspergillus Nidulans and Secretion of Mature Interleukin-6. Nat Biotechnol 9, 378–381 (1991). https://doi.org/10.1038/nbt0491-378

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