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:

High-Level Direct Expression of Semi-Synthetic Human Interleukin-6 in Escherichia coli and Production of N-Terminus Met-Free Product

Bio/Technology volume 8, pages 1036–1040 (1990)Cite this article

Abstract

We have developed a direct expression system for high-level production of recombinant human interleukin-6 (rhIL-6) in Escherichia coli. In this system, (i) the natural N-terminal coding region of the hIL-6 gene was replaced by a synthetic sequence containing A-T rich codons, (ii) dual Shine-Dalgarno (SD) sequences were employed, (iii) an A-T rich segment was inserted in front of the initiation codon to avoid putative mRNA secondary structure in the region and (iv) the natural amber termination codon of the hIL-6 gene was changed to an ocher stop codon. The hlL-6 polypeptide, synthesized at a high level, formed cytoplasmic inclusion bodies. After refolding, the N-terminal methionine was removed by aminopeptidase-P in vitro. The purified recombinant hIL-6 had B-cell differentiation activity equivalent to natural IL-6 from a human T-cell culture.

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. Hirano, T., Yasukawa, K., Harada, H., Taga, T., Watanabe, Y., Matsuda, T., Kashiwamura, S., Matsui, H., Takahara, Y., Taniguchi, T. and Kishimoto, T. 1986. Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin. Nature 324: 73–76.

    Article  CAS  Google Scholar 

  2. Hirano, T. and Kishimoto, T. 1989. Interleukin-6 (IL-6). In: Handbook of Experimental Pharmacology: “Peptide Growth Factors and Their Receptors”. Sporn, M. B. and Roberts, A. B. (Eds.). Springer-Verlag, Berlin (In press).

    Google Scholar 

  3. Ishibashi, T., Kimura, H., Shikama, Y., Uchida, T., Kariyone, S., Hirano, T., Kishimoto, T., Takatsuki, F. and Akiyama, Y. 1989. Interleukin-6 is a potent thrombopoietic factor in vivo in mice. Blood 74: 1241–1244.

    CAS  Google Scholar 

  4. Jambou, R.C., Snouwaert, J.N., Bishop, G.A., Stebbins, J.R., Fre-linger, J.A. and Fowlkes, D.M. 1988. High-level expression of a bioengineered, cysteine-free hepatocyte-stimulating factor (interleukin 6)-like protein. Proc. Natl. Acad. Sci. USA 85: 9426–9430.

    Article  CAS  Google Scholar 

  5. Asagoe, Y., Yasukawa, K., Saito, T., Maruo, N., Miyata, K., Kono, T., Miyake, T., Kato, T., Kakidani, H. and Mitani, M. 1988. Human B-cell stimulatory factor-2 expressed in Escherichia coli. Bio/Technology 6: 806–809.

    CAS  Google Scholar 

  6. Yoshimoto, T., Tone, H., Honda, T., Osatomi, K., Kobayashi, R. and Tsuru, D. 1989. Sequencing and high expression of aminopeptidase P gene from Escherichia coli HB101. J. Biochem. 105: 412–416.

    Article  CAS  Google Scholar 

  7. Bolivar, F. and Backman, K. 1979. Plasmids of Escherichia coli as cloning vectors. Methods Enzymol. 68: 245–267.

    Article  CAS  Google Scholar 

  8. Ikemura, T. and Ozeki, H. 1982. Codon usage and transfer RNA contents: organism-specific codon-choice patterns in reference to the isoacceptor contents. Cold Spring Harbor Symp. Quant. Biol. 47: 1087–1097.

    Article  CAS  Google Scholar 

  9. Hall, M.N., Gabay, J., Debarbouille, M. and Schwanz, M. 1982. A role for mRNA secondary structure in the control of translation initiation. Nature 295: 616–618.

    Article  CAS  Google Scholar 

  10. Wood, C.R., Boss, M.A., Patel, T.P. and Emtage, J.S. 1984. The influence of messenger RNA secondary structure on expression of an immunoglobulin heavy chain in Escherichia coli. Nucleic Acids Res. 12: 3937–3950.

    Article  CAS  Google Scholar 

  11. Tessier, L., Sondermeyer, P., Faure, T., Dreyer, D., Benavente, A., Villeval, D., Courtney, M. and Lecocq, J. 1984. The influence of mRNA primary and secondary structure on human IFN-γ gene expression in E. coli. Nucleic Acids Res. 12: 7663–7675.

    Article  CAS  Google Scholar 

  12. Saiki, O. and Ralph, P. 1983. Clonal differences in response to T cell replacing factor (TRF) for IgM secretion and TRF receptors in a human B lymphoblast cell line. Eur. J. Immunol. 13: 31–44.

    Article  CAS  Google Scholar 

  13. Tonouchi, N., Oouchi, N., Kashima, N., Kawai, M., Nagase, K., Okano, A., Matsui, H., Yamada, K., Hirano, T. and Kishimoto, T. 1988. High-level expression of human BSF-2/IL-6 cDNA in Escherichia coli using a new type of expression-preparation system. J. Biochem. 104: 30–34.

    Article  CAS  Google Scholar 

  14. Hui, A., Hayflick, J., Dinkelspiel, K. and de Boer, H.A. 1984. Mutagenesis of the three bases preceding the start codon of the β-galactosidase mRNA and its effect on translation in Escherichia coli. EMBO J. 3: 623–629.

    Article  CAS  Google Scholar 

  15. Looman, A.C., Bodlaender,J., Comstock, L.J., Eaton, D., Jhurani, P., de Boer, H.A. and van Knippenberg, P.H. 1987. Influence of the codon following the ATG initiator codon on the expression of a modified lacZ gene in Escherichia coli. EMBO J. 6: 2489–2492.

    Article  CAS  Google Scholar 

  16. Schoner, B.E., Hsiung, H.M., Belagaje, R.M., Mayne, N.G. and Schoner, R.G. 1984. Role of mRNA translational efficiency in bovine growth hormone expression in Escherichia coli. Proc. Natl. Acad. Sci. USA 81: 5403–5407.

    Article  CAS  Google Scholar 

  17. Schoner, B.E., Belagaje, R.M. and Schoner, R.G. 1986. Translation of a synthetic two-cistron mRNA in Escherichia coli. Proc. Natl. Acad. Sci. USA 83: 8506–8510.

    Article  CAS  Google Scholar 

  18. Bonekamp, F., Dalbøge, H., Christensen, T. and Jensen, K.F. 1989. Translation rates of individual codons are not correlated with tRNA abundances or with frequencies of utilization in Escherichia coli. J. Bacteriol. 171: 5812–5816.

    Article  CAS  Google Scholar 

  19. Brakenhoff, J.J., Hart, M. and Aarden, L.A. 1989. Analysis of human IL-6 mutants expressed in Escherichia coli. Biologic activities are not affected by deletion of amino acids 1–28. J. Immunol. 143: 1175–1182.

    CAS  PubMed  Google Scholar 

  20. Murata, M., Eto, Y., Shibai, H., Sakai, M. and Muramatsu, M. 1988. Erythroid differentiation factor is encoded by the same mRNA as that of the inhibin βA chain. Proc. Natl. Acad. Sci. USA 85: 2434–2438.

    Article  CAS  Google Scholar 

  21. Ben-Bassat, A., Bauer, K., Chang, S., Myambo, K., Boosman, A. and Chang, S. 1987. Processing of the initiation methionine from proteins: properties of the Escherchia coli methionine aminopeptidase and its gene structure. J. Bacteriol. 169: 751–757.

    Article  CAS  Google Scholar 

  22. Ben-Bassat, A. and Bauer, K. 1987. Amino-terminal processing of proteins. Nature 326: 315.

    Article  Google Scholar 

  23. Towbin, H., Staehelin, T. and Gordon, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76: 4350–4354.

    Article  CAS  Google Scholar 

Download references

Author information

Author notes

  1. Hisashi Yasueda: Corresponding author.

Authors and Affiliations

  1. Central Research Laboratories, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki, 210, Japan

    Hisashi Yasueda, Kazuo Nagase, Akira Hosoda, Yukio Akiyama & Kazuhiko Yamada

Authors
  1. Hisashi Yasueda
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kazuo Nagase
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Akira Hosoda
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yukio Akiyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kazuhiko Yamada
    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

Yasueda, H., Nagase, K., Hosoda, A. et al. High-Level Direct Expression of Semi-Synthetic Human Interleukin-6 in Escherichia coli and Production of N-Terminus Met-Free Product. Nat Biotechnol 8, 1036–1040 (1990). https://doi.org/10.1038/nbt1190-1036

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1190-1036

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