Skip to main content
SpringerLink
Log in

A rapid method for solubilization of chimeric human interferon regulatory factor-1 (IRF-1) protein in Escherichia coli

  • Original Article
  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Interferon regulatory factor-1 (IRF-1) is a vertebrate transcription factor that plays significant roles in cell cycle regulation, anti-viral response, tumor suppression and immune response. High-level expression of recombinant IRF-1 at 37 °C leads to the formation of insoluble aggregates (insoluble fraction) in Escherichia coli (E. coli), which usually devoid of biological activity. In this study, we use chemical additives such as mannitol, proline, l-arginine and CTAB (cetyl trimethly ammonium bromide) at the recommended concentration during cell lysis to aid in solubility at 37 °C. The use of additives resulted in the increased solubility of the recombinant glutathione S-transferase-linked human IRF-1, with l-arginine being most effective. Here, we developed an efficient process for the manufacturing of soluble IRF-1 with the aid of minimizing the formation of degradation products and optimizing protein purification conditions. This result was further confirmed by western blot with anti-GST and anti-IRF-1 polyclonal antibodies. The functionality of GST-huIRF-1 was attained by elerophoretic mobility shift assay study as a clear band shifting showed with virus response element-Interferon beta (VRE-IFNβ) promoter region. Taken together, the biological activity of purified GST-huIRF-1 was also optimized and confirmed by supershift assay concluded that GST-huIRF-1 interacts with the VRE motif of IFNβ promoter that reflected to require for IFNβ gene regulation. We describe a straightforward approach for the production of absolutely soluble and biologically active IRF-1 in E. coli. This method can be further used for the study of other recombinant proteins and this study will pave way for the analysis of IRF-1 function in vitro.

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

Similar content being viewed by others

References

  1. Alsamman K, El-Masry OS (2018) Interferon regulatory factor 1 inactivation in human cancer. Biosci Rep 38(3):BSR20171672

    Article  CAS  Google Scholar 

  2. Tamura T, Yanai H, Savitsky D, Taniguchi T (2008) The IRF family transcription factors in immunity and oncogenesis. Annu Rev Immunol 26:535–584

    Article  CAS  Google Scholar 

  3. Tanaka N, Ishihara M, Lamphier MS, Nozawa H, Matsuyama T, Mak TW, Taniguchi T (1996) Cooperation of the tumour suppressors IRF-1 and p53 in response to DNA damage. Nature 382(6594):816–818

    Article  CAS  Google Scholar 

  4. Lee SH, Kim JW, Lee HW, Cho YS, Oh SH, Kim YJ, Lee JH (2003) Interferon regulatory factor-1 (IRF-1) is a mediator for interferon-γ induced attenuation of telomerase activity and human telomerase reverse transcriptase (hTERT) expression. Oncogene 22(3):381–391

    Article  CAS  Google Scholar 

  5. Shao L, Hou W, Scharping NE, Vendetti FP, Srivastava R, Roy CN, Menk AV, Wang Y, Chauvin JM, Karukonda P, Thorne SH (2019) IRF1 inhibits antitumor immunity through the upregulation of PD-L1 in the tumor cell. Cancer Immunol Res 7(8):1258–1266

    Article  CAS  Google Scholar 

  6. MacDonald NJ, Kuhl D, Maguire D, Näf D, Gallant P, Goswamy A, de la Fuente J (1990) Different pathways mediate virus inducibility of the human IFN-α1 and IFN-β genes. Cell 60(5):767–779

    Article  CAS  Google Scholar 

  7. Mishra SK, Kumar N, Kumar G, Kashav T, Prakash K (2018) Recombinant human interferon regulatory factor-1 (IRF-1) protein expression and solubilisation study in Escherichia coli. Mol Biol Rep 45(5):1367–1374

    Article  Google Scholar 

  8. Upreti M, Rath PC (2005) Expression and DNA binding activity of the recombinant interferon regulatory factor-1 (IRF-1) of mouse. Mol Biol Rep 32(2):103–116

    Article  CAS  Google Scholar 

  9. Prakash K, Rath PC (2012) Mouse interferon regulatory factor-2: expression, purification and DNA binding activity. Mol Biol Rep 39(1):599–606

    Article  CAS  Google Scholar 

  10. Baneyx F, Mujacic M (2004) Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol 22(11):1399–1408

    Article  CAS  Google Scholar 

  11. Francis DM, Page R (2010) Strategies to optimize protein expression in E. coli. Curr Protoc Protein Sci 61(1):5.24.1–29

    Article  Google Scholar 

  12. Bondos SE, Bicknell A (2003) Detection and prevention of protein aggregation before, during, and after purification. Anal Biochem 316(2):223–231

    Article  CAS  Google Scholar 

  13. Leibly DJ, Nguyen TN, Kao LT, Hewitt SN, Barrett LK, Van Voorhis WC (2012) Stabilizing additives added during cell lysis aid in the solubilization of recombinant proteins. PLoS One 7(12):e52482

    Article  CAS  Google Scholar 

  14. Harada H, Willison K, Sakakibara J, Miyamoto M, Fujita T, Taniguchi T (1990) Absence of the type I IFN system in EC cells: transcriptional activator (IRF-1) and repressor (IRF-2) genes are developmentally regulated. Cell 63(2):303–312

    Article  CAS  Google Scholar 

  15. Karki R, Sharma BR, Banoth B, Malireddi RS, Samir P, Tuladhar S, Mummareddy H, Burton AR, Vogel P, Kanneganti TD (2020) Interferon regulatory factor 1 regulates PANoptosis to prevent colorectal cancer. JCI Insight 5(12):e136720

    Article  Google Scholar 

  16. Gupta A, Rath PC (2012) Expression, purification and characterization of the interferon-inducible, antiviral and tumour-suppressor protein, human RNase L. J Biosci 37(1):103–113

    Article  CAS  Google Scholar 

  17. Reddy KRC, Lilie H, Rudolph R, Lange C (2005) L-Arginine increases the solubility of unfolded species of hen egg white lysozyme. Protein Sci 14(4):929–935

    Article  Google Scholar 

  18. Nakagawa K, Yokosawa H (2000) Degradation of transcription factor IRF-1 by the ubiquitin–proteasome pathway: the C-terminal region governs the protein stability. Eur J Biochem 267(6):1680–1686

    Article  CAS  Google Scholar 

  19. Bandyopadhyay A, Saxena K, Kasturia N, Dalal V, Bhatt N, Rajkumar A, Chakraborty K (2012) Chemical chaperones assist intracellular folding to buffer mutational variations. Nat Chem Biol 8(3):238–245

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The researchers thank current and past members of the Central University of South Bihar for helpful discussions and research fellowships from the UGC to Santosh Kumar Mishra.

Author information

Authors and Affiliations

  1. Recombinant DNA Technology Laboratory, Department of Biotechnology, Central University of South Bihar, Gaya, 824236, India

    Santosh Kumar Mishra, Ruchi Verma, Priyanka Kumari, Anjali Kumari & Krishna Prakash

Authors
  1. Santosh Kumar Mishra
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ruchi Verma
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Priyanka Kumari
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anjali Kumari
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Krishna Prakash
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Krishna Prakash.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants performed by any of the authors.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mishra, S.K., Verma, R., Kumari, P. et al. A rapid method for solubilization of chimeric human interferon regulatory factor-1 (IRF-1) protein in Escherichia coli. Mol Biol Rep 48, 2307–2314 (2021). https://doi.org/10.1007/s11033-021-06260-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-021-06260-w

Keywords

Search

Navigation