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Expression, refolding, and characterization of GFE peptide-fused human interferon-α2a in Escherichia coli

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Abstract

Interferon-α2a (IFN-α2a) has been used for the treatment of various viral infections and cancers for many years. However some untolerable side effects have limited its application in some aspects. To evaluate whether or not an oligopeptide containing GFE motif can home human IFN-α2a to specific tissues, a fusion gene was constructed by fusing the coding sequence of GFE peptide (CGFECVRQCPERC), which was screened from phage display peptide library, to the 3′ end of human IFN-α2a gene by recombinant DNA technique. Fusion protein rhIFN-α2a-GFE was expressed in Escherichia coli as inclusion bodies using a T7 RNA polymerase expression system, pET-22b, refolded through dialysis and purified to homogeneity to >95% of purity by affinity chromatography. Characterization by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting demonstrated the authenticity of the fusion protein. Purified rhIFN-α2a-GFE was found to be functionally active in terms of its antiviral activity for about 2.5×108 IU/mg in vitro. Yields of the purified fusion protein were about 200 mg/L of culture medium. Tissue distribution assay in mouse showed that at 30 min IFN-α2a could be enriched sevenfold higher in lung in the targeted IFN group of mice than in the standard IFN group of mice, and last for a long time. At 1 h, IFN-α2a in the targeted IFN group was still 4.02-fold higher than that in the standard group. This confirmed that GFE peptide has the ability to selectively deliver its fusion partner IFN-α2a to lungs. The results also showed that the IFN-α2a-GFE could be specifically enriched in kidney and liver. Its distribution in kidney was concordant with the finding of GFE receptor, MDP, in kidney. However, the IFN-α2a-GFE in liver may imply some significance in pharmacology and toxicology.

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References

  1. Isaacs, A. and Lindenmann, J. (1957), Proc. R. Soc. London Ser. B 147, 258–267.

    Article  CAS  Google Scholar 

  2. Aguet, M., Grobke, M., and Dreiding, P. (1984), Virology 132, 211–216

    Article  CAS  Google Scholar 

  3. Meager, A. (2002), J. Immunol. Methods 261, 21–36.

    Article  CAS  Google Scholar 

  4. Pfeffer, L. M., Dinarello, C. A., Herberman, R. B., Williams, B. R., Borden, E. C., Bordens, R., Walter, M. R., et al. (1998), Cancer Res. 58, 2489–2499.

    CAS  Google Scholar 

  5. Sen, G. C. and Lengyel, P. (1992), J. Biol. Chem. 267, 5017–5020.

    CAS  Google Scholar 

  6. Samuel, C. E. (2001), Clin. Microbiol. Rev. 14, 778–809.

    Article  CAS  Google Scholar 

  7. Lauer, G. M. and Walker, B. D. (2001), N. Engl. J. Med. 345, 41–52.

    Article  CAS  Google Scholar 

  8. Samuel, C.E. (1991), Virology 18, 31–11.

    Google Scholar 

  9. Jonasch, E. and Haluska, F. G. (2001), Oncologist 6, 34–55.

    Article  CAS  Google Scholar 

  10. Vilcek, J. and Sen, G. C. (1996), in Fundamental Virology, 3rd ed (Fields, B. N., Knipe, D. M., and Howley, P. M., eds.), Lippincot-Raven, Philadelphia, pp. 341–365.

    Google Scholar 

  11. Motzer, R. J., Bacik, J., Murphy, B. A., Russo, P., and Mazumdar, M. (2002), J. Clin. Oncol. 20, 289–296.

    Article  CAS  Google Scholar 

  12. Mahon, F. X., Delbrel, X., Cony-Makhoul, P., Faberes, C., Boiron, J. M., Barthe, C., Bilhou-Nabera, C., et al. (2002), J. Clin. Oncol., 20, 214–220.

    Article  CAS  Google Scholar 

  13. Stark, J. J., Dillman, R. O., Schulof, R., Wiemann, M. C., Barth, N. M., Honeycutt, P. J., and Soori, G. (1998), Cancer 82, 1677–1681.

    Article  CAS  Google Scholar 

  14. Rajotte, D., Arap, W., Hagedorn, M., Koivunen, E., Pasqualini, R., and Ruoslahti, E. (1998), J. Clin. Invest. 102, 430–437.

    CAS  Google Scholar 

  15. Weissmann, C. and Weber, H. (1986), Prog. Nucleic Acid Res. Mol. Biol. 33, 251–300.

    CAS  Google Scholar 

  16. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951), J. Biol. Chem. 193, 265–275.

    CAS  Google Scholar 

  17. Familletti, P. C., Rubinstein, S., and Pestka, S. (1981), Methods Enzymol. 78, 387–394.

    Article  CAS  Google Scholar 

  18. Standardization Committee of Department of Health in China. (2000), in Chinese Regulation for Biological Products (Pan, Z. A., Zhang, X. H., and Duan, Z. B., eds.), Chinese Chemical Industry Press, Beijing, China, pp. 373–375.

    Google Scholar 

  19. Rajotte, D. and Ruoslahti, E. (1999), J. Biol. Chem. 274, 11,593–11,598.

    Article  CAS  Google Scholar 

  20. Thatcher, D. R. and Panayotatos, N. (1996), Methods Enzymol. 119, 166–177.

    Google Scholar 

  21. Swaminathan, S. and Khanna, N. (1999), Protein Express. Purif. 15, 236–242.

    Article  CAS  Google Scholar 

  22. Neves, F. O., Ho, P. L., Raw, I., Pereira, C. A., Moreira, C., and Nascimento, A. L. (2004), Protein Express. Purif. 35, 353–359.

    Article  CAS  Google Scholar 

  23. Keynan, S., Hooper, N. M., and Turner, A. J. (1996), in Zinc Metalloproteases in Health and Disease, Hooper, N. M., ed., Taylor & Francis, London, pp. 285–309.

    Google Scholar 

  24. Yamaya, M., Sekizawa, K., Terajima, M., Okinaga, S., Ohrui, T., and Sasaki, H. (1998), Respir. Physiol. 111, 101–109.

    Article  CAS  Google Scholar 

  25. Hirota, T., Nishikawa, Y., Tanaka, M., Igarashi, T., and Kitagawa, H. (1986), Eur. J. Biochem. 160, 521–525.

    Article  CAS  Google Scholar 

  26. Pytela, R., Pierschbacher, M. D., and Ruoslahti, E. (1985), Cell 40, 191–198.

    Article  CAS  Google Scholar 

  27. Habib, G. M., Shi, Z. Z., Cuevas, A. A., Guo, Q., Matzuk, M. M., and Lieberman, M. W. (1998), Proc. Natl. Acad. Sci. USA 95, 4859–4863.

    Article  CAS  Google Scholar 

  28. Habib, G. M., Barrios, R., Shi, Z. Z., and Lieberman, M. W. (1996), J. Biol. Chem. 271, 16,273–16,280.

    CAS  Google Scholar 

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Authors and Affiliations

  1. Biotechnology Center of Fourth Military Medical University, Chang Le Xi Lu #17, 710032 Shaanxi, Xi'an, People's Republic of China

    Zhen Yan, Jihong Shi, Chunjie Bao, Wei Han & Yingqi Zhang

  2. State Key Laboratory of Cancer Biology, Chang Le Xi Lu #17, 710032, Shaanxi, Xi'an, People's Republic of China

    Zhen Yan, Jihong Shi, Chunjie Bao, Wei Han & Yingqi Zhang

  3. College of Pharmaceutical Science of Lanzhou University, Key Laboratory of Preclinical Study for New Traditional Chinese Medicine of Gansu Province, Lanzhou University, 730000, Gansu, Lanzhou, People's Republic of China

    Li Lu & Yongjie Wu

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  1. Zhen Yan
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  2. Li Lu
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Correspondence to Yingqi Zhang.

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Yan, Z., Lu, L., Shi, J. et al. Expression, refolding, and characterization of GFE peptide-fused human interferon-α2a in Escherichia coli . Appl Biochem Biotechnol 133, 149–162 (2006). https://doi.org/10.1385/ABAB:133:2:149

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  • DOI: https://doi.org/10.1385/ABAB:133:2:149

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