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.
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References
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.
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).
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.
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.
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.
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.
Bolivar, F. and Backman, K. 1979. Plasmids of Escherichia coli as cloning vectors. Methods Enzymol. 68: 245–267.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Ben-Bassat, A. and Bauer, K. 1987. Amino-terminal processing of proteins. Nature 326: 315.
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.
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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
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DOI: https://doi.org/10.1038/nbt1190-1036