Abstract
A genetic approach to facilitate large–scale production and down stream processing of heterologous proteins expressed in bacteria is described. The gene is fused after a synthetic fragment encoding two IgG–binding domains derived from staphylococcal protein A. The fusion product is secreted to the growth medium of Escherichia coli, and purified using the IgG–binding moiety as affinity “tail”. We demonstrate that the expression system can be used for the production of biologically active human insulin–like growth factor I in a 1000 litre scale. The process includes fermentation, cell separation, affinity chromatography on IgG Sepharose Fast Flow and site specific chemical cleavage of the fusion protein.
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Ross, M.J. 1981. Production of medically important polypeptides using recombinant DNA technology, p. 33–48. In: Insulins, Growth Hormones and Recombinant DNA Technology. J.L. Gueriguian (ed) Raven Press, New York.
Nilsson, B., Abrahmsen, L. and Uhlen, M. 1985. Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors. EMBO J. 4: 1075–1080.
Uhlen, M., Nilsson, B., Guss, B., Lindberg, M., Gatenbeck, S. and Philipson, L. 1983. Gene fusion vectors based on the gene for staphylococcal protein A. Gene 23: 369–378.
Nilsson, B., Holmgren, E., Josephson, S., Gatenbeck, S., Philipson, L. and Uhlen, M. 1985. Efficient secretjon and purification of human insulin-like growth factor I with a gene fusion vector in Staphylococci. Nucl. Acids Res. 13: 1151–1162.
Germino, J. and Bastia, D. 1984. Rapid purification of a cloned gene product by genetic fusion and site specific proteolysis. Proc. Natl. Acad. Sci. USA 81: 4692–4696.
Ullman, A. 1984. One-step purification of hybrid proteins which have beta-galactosidase activity. Gene 29: 27–31.
Sassenfeld, H.M. and Brewer, S.J. 1984. A polypeptide fusion designed for the purification of recombinant proteins. Bio/Technology 2: 76–81.
Nilsson, B., Moks, T., Jansson, B., Abrahmsen, L., Elmblad, A., Holmgren, E., Henrichson, C., Jones, A. and Uhlen, M. 1987. Engineering of the IgG binding region of staphylococcal protein A. Protein Engineering, in press.
Abrahmsen, L., Moks, T., Nilsson, B. and Uhlen, M. 1986. Secretion of human insulin-like growth factor I to the culture medium of Escherichia coli. Nucl. Acids Res. 14: 7487–7500.
Humbel, R.E. 1984. Insulin-like growth factors, somatomedins, and multiplication stimulating activity chemistry, p. 57–79. In: Hormonal Proteins and Peptides. Vol XII. C. H. Li (ed.), Academic Press, New York.
Moks, T., Abrahmsen, L., Holmgren, E., Billich, M., Olsson, A., Pohl, G., Sterky, C., Hultberg, H., Josephson, S., Holmgren, A., Jörnvall, H., Uhlen, M. and Nilsson, B. 1987. Bacterial production of human insulin-like growth factor I—use of optimized gene fusion vectors that facilitate protein purification. Proc. Natl. Acad. Sci., USA, submitted.
Moks, T., Abrahmsen, L., Nilsson, B., Hellman, U., Sjøquist, J. and Uhlen, M. 1986. Staphylococcal protein A consists of five IgG-binding domains. Eur. J. Biochem. 156: 637–643.
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Moks, T., Abrahmsén, L., Österlöf, B. et al. Large–Scale Affinity Purification of Human Insulin–Like Growth Factor I from Culture Medium of Escherichia Coli. Nat Biotechnol 5, 379–382 (1987). https://doi.org/10.1038/nbt0487-379
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DOI: https://doi.org/10.1038/nbt0487-379
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