Skip to main content
SpringerLink
Log in

Soluble Expression, Rapid Purification and Antiviral Activity of Recimbinant Bovine Interferon-α in Escherichia coli

  • Published:
Applied Biochemistry and Microbiology Aims and scope Submit manuscript

Abstract

In this study, the cDNA of mature bovine interferon-α (BoIFN-α) gene was cloned and expressed in the Escherichia coli expression system. Total RNA was extracted from bovine liver cells and reverse transcribed into cDNA, and then BoIFN-α gene fragment was amplified with specific primers by PCR method before being ligated into the pET-32a (+) expression vector. Since the pET-32a (+) plasmid expresses the thioredoxin A (TrxA) tag protein, it can increase the solubility of the target protein. In order to express the target protein, the pET-32a-BoIFN-α recombinant plasmid was transformed into E. coli Rosetta (DE3) strain followed by IPTG induction. It was found that the recombinant fusion protein Trx-rBoIFN-α was finally expressed in a soluble form in the host cell, accounting for about 33.45% of the total cellular protein. The protein was purified by two-step chromatography, including Ni2+-chelating Sepharose affinity chromatography and DEAE anion exchange chromatography. The purity of the purified product reached 93.0%, and the cytopathic effects (CPE) inhibition method was adopted to determine the antiviral activity of the expressed fusion protein in vesicular stomatitis virus/ Madin-Darby bovine kidney cells titration system. The specific activity of the recombinant fusion protein Trx-rBoIFN-α was measured as (3.6 ± 0.25) × 106 U/mg. After cutting of the Trx tag protein by enterokinase digestion, the remaining solo protein was confirmed as rBoIFN-α protein by Western blot assay. These findings will enable us to produce a large number of bioactive rBoIFN-α protein for future application.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.

Similar content being viewed by others

REFERENCES

  1. Tomasello, E., Pollet, E., Vu Manh, T.P., Uze, G., and Dalod, M., Front Immunol., 2014, vol. 5, p. 526.

    Article  Google Scholar 

  2. Maher, S.G., Romero-Weaver, A.L., Scarzello, A.J., and Gamero, A.M., Curr. Med. Chem., 2007, vol. 14, no. 12, pp. 1279–1289.

    Article  CAS  Google Scholar 

  3. Le Bon, A., Schiavoni, G., D’Agostino, G., Gresser, I., Belardelli, F., and Tough, D.F., Immunity, 2001, vol. 14, no. 4, pp. 461–470.

    Article  CAS  Google Scholar 

  4. Sainz, B.Jr. and Halford, W.P., J. Virol., 2002, vol. 76, no. 22, pp. 11541–11550.

    Article  CAS  Google Scholar 

  5. Yao, Q., Qian, P., Cao, Y., He, Y., Si, Y., Xu, Z., and Chen, H., Eur. Cytokine Netw., 2007, vol. 18, no. 2, pp. 71–77.

    CAS  PubMed  Google Scholar 

  6. Moraes, M.P., de Los Santos, T., Koster, M., Turecek, T., Wang, H., Andreyev, V.G., and Grubman, M.J., J. Virol., 2007, vol. 81, no. 13, pp. 7124–7135.

    Article  CAS  Google Scholar 

  7. Diaz-San Segundo, F., Weiss, M., Perez-Martin, E., Koster, M.J., Zhu, J., Grubman, M.J., and de los Santos, T., Virology, 2011, vol. 413, no. 2, pp. 283–292.

    Article  CAS  Google Scholar 

  8. Gillespie, J., Scott, F., Geissinger, C., and Schiff, E., Zentralbl. Veterinarmed B, 1986, vol. 33, no. 10, pp. 771–776.

    CAS  PubMed  Google Scholar 

  9. Gillespie, J.H., Scott, F.W., Geissinger, C.M., Czarniecki, C.W., and Scialli, V.T., J. Clin. Microbiol., 1986, vol. 24, no. 2, pp. 240–244.

    Article  CAS  Google Scholar 

  10. Van Wyk, B., Snider, M., Scruten, E., Littel-van den Hurk, S.V., and Napper, S., Vet. Microbiol., 2016, vol. 195, pp. 104–114.

    Article  CAS  Google Scholar 

  11. Chinsangaram, J., Piccone, M.E., and Grubman, M.J., J. Virol., 1999, vol. 73, no. 12, pp. 9891–9898.

    Article  CAS  Google Scholar 

  12. Ohmann, H.B., Gilchrist, J.E., and Babiuk, L.A., J. Gen. Virol., 1984, vol. 65, part 9, pp. 1487–1495.

    Article  CAS  Google Scholar 

  13. Shao, J.W., Cao, C., Bao, J., Liu, H.T., Peng, T.Q., Gao, M.C., and Wang, J.W., J. Interf. Cytok. Res., 2015, vol. 35, no. 3, pp. 168–175.

    Article  CAS  Google Scholar 

  14. Akiyama, K., Sugii, S., and Hirota, Y., J. Vet. Med. Sci., 1993, vol. 55, no. 3, pp. 449–452.

    Article  CAS  Google Scholar 

  15. Liu, Z.Q. and Yang, P.C., N. Am. J. Med. Sci., 2012, vol. 4, no. 12, pp. 651–655.

    Article  Google Scholar 

  16. Mahmoudi, S., Abtahi, H., Bahador, A., Mosayebi, G., Salmanian, A.H., and Teymuri, M., Maedica (Buchar), 2012, vol. 7, pp. 241–246.

    Google Scholar 

  17. Kruger, N.J., Methods Mol. Biol., 1994,vol. 32, pp. 9–15.

    CAS  PubMed  Google Scholar 

  18. Tu, Y.B., Wang, G., Wang, Y.Q., Chen, W.Y., Zhang, L., Liu, Y.G., et al., Microbiol. Res., 2016, vol. 191, pp. 12–18.

    Article  CAS  Google Scholar 

  19. Chiba, K., Kawakami, K., Tohyama, K., Toxicol. In. Vitro., 1998, vol. 12, no.3, pp.251–258.

    Article  CAS  Google Scholar 

  20. Liu, K., Liao, X., Zhou, B., Yao, H., Fan, S., Chen, P., and Miao, D., Res. Vet. Sci., 2013, vol. 95, no. 3, pp. 950–956.

    Article  CAS  Google Scholar 

  21. Reed, L. and Muench, H., Am. J. Hyg., 1938, vol. 27, pp. 493–497.

    Google Scholar 

  22. Hou, F., Liu, K., Shen, T., Zhou, B., Cao, R., Li, P., and Chen, P., Res. Vet. Sci., 2011, vol. 91, no. 3, pp. e73–e79.

    Article  CAS  Google Scholar 

  23. Ruttanapumma, R., Nakamura, M., and Takehara, K., J. Vet. Med. Sci., 2005, vol. 67, no. 1, pp. 25–28.

    Article  CAS  Google Scholar 

  24. Marcus, P.I., van der Heide, L., and Sekellick, M.J., J. Interf. Cytok. Res., 1999, vol. 19, no. 8, pp. 881–885.

    Article  CAS  Google Scholar 

  25. Schultz, U., Rinderle, C., Sekellick, M.J., Marcus, P.I., and Staeheli, P., Eur. J. Biochem., 1995, vol. 229, no. 1, pp. 73–76.

    Article  CAS  Google Scholar 

  26. Chowdhury, A., Shah, S., Babu, S., Rai, R.R., Prasad, V.G., Rao, R., et al., Trop Gastroenterol., 2005, vol. 26, no. 2, pp. 70–72.

    PubMed  Google Scholar 

  27. Zhou, X., Song, Z., Liu, X., Jia, F., and Wang, Y., Appl. Biochem. Biotechnol., 2011, vol. 163, no. 8, pp. 981–993.

    Article  CAS  Google Scholar 

  28. Charlton, A. and Zachariou, M., Methods Mol. Biol., 2008, vol. 421, pp. 137–149.

    CAS  PubMed  Google Scholar 

  29. Neves, F.O., Ho, P.L., Raw, I., Pereira, C.A., Moreira, C., and Nascimento, A.L., Protein Expr. Purif., 2004, vol. 35, no. 2, pp. 353–359.

    Article  CAS  Google Scholar 

  30. Manual of Clinical Laboratory Immunology, Rose N.R, Friedman F.H., Fahey, J.L., Eds., 1986, Washington, DC: American Society for Microbiology, pp. 295–299.

    Google Scholar 

  31. Rubinstein, S., Familletti, P.C., and Pestka, S., J. Virol., 1981, vol. 37, no. 2, pp. 755–758.

    Article  CAS  Google Scholar 

  32. Hawkins, M.J., Borden, E.C., Merritt, J.A., Edwards, B.S., Ball, L.A., Grossbard, E., and Simon, K.J., J. Clin. Oncol., 1984, vol. 2, no. 3, pp. 221–226.

    Article  CAS  Google Scholar 

  33. McManus, N.H., Appl. Environ. Microbiol., 1976, vol. 31, no. 1, pp. 35–38.

    Article  CAS  Google Scholar 

  34. Armstrong, J.A., Methods Enzymol., 1981, vol. 78, Pt. A, pp. 381–387.

  35. Samuel, C.E., Clin. Microbiol. Rev., 2001, vol. 14, no. 4, pp. 778–809.

    Article  CAS  Google Scholar 

  36. Zhao, J., Yu, H.Y., Zhao, Y., Li, F.H., Zhou, W., Xia, B.B., et al., Prep. Biochem. Biotechnol., 2019, vol. 49, no.2, pp.192–201.

    Article  CAS  Google Scholar 

Download references

ACKNOWLEDGMENTS

The authors are grateful to all the Research Staff in Wuhu Interferon Bio-products Industry Research Institute Co., Ltd (Wuhu, Anhui, P.R. China) for technical assistance.

Funding

This research was funded by the programs from the National key R&D Program of China (Grant nos. 2017YFD0500906 and 2017YFD0501000), the Natural Science Foundation of Anhui Province (Grant no. 1808085MC75), Scientific research activities of postdoctoral researchers in Anhui (Grant no. 2017B194).

Author information

Authors and Affiliations

  1. Department of Microbiology, Anhui Medical University, 230032, Hefei, Anhui Province, P.R. China

    H.-Y. Yu, M.-L. Wang & J. Zhao

  2. Wuhu Interferon Bio-products Industry Research Institute Co., Ltd., 241000, Wuhu, Anhui Province, P.R. China

    J. Liu, Z.-Y. He, W. Zhou, B.-B. Xia, M. Wang, M.-L. Wang & J. Zhao

  3. Department of Pathology and Cell Biology, Columbia University, 10032, New York, USA

    J. Chen

  4. Dalian SanYi animal medicine Co., Ltd., 116036, Dalian, Liaoning Province, P.R. China

    G.-T. Jiang

  5. Wuhu Overseas Students Pioneer Park, 241000, Wuhu, Anhui Province, P.R. China

    J. Zhao

Authors
  1. H.-Y. Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J. Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Z.-Y. He
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. W. Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. B.-B. Xia
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. M. Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. J. Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. M.-L. Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. G.-T. Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. J. Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to M.-L. Wang, G.-T. Jiang or J. Zhao.

Ethics declarations

The authors declare that they have no conflict of interest. This article does not contain any studies with human participants or animals performed by any of the authors.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yu, HY., Liu, J., He, ZY. et al. Soluble Expression, Rapid Purification and Antiviral Activity of Recimbinant Bovine Interferon-α in Escherichia coli. Appl Biochem Microbiol 56, 154–163 (2020). https://doi.org/10.1134/S0003683820020143

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S0003683820020143

Keywords:

Search

Navigation