Skip to main content
SpringerLink
Log in

Recombinant Production and Characterization of SAC, the Core Domain of Par-4, by SUMO Fusion System

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

Abstract

Prostate apoptosis response-4 (Par-4), an anticancer protein that interacts with cell surface receptor GRP78, can selectively suppress proliferation and induce apoptosis of cancer cells. The core domain of Par-4 (aa 137–195), designated as SAC, is sufficient to inhibit tumor growth and metastasis without harming normal tissues and organs. Nevertheless, the anticancer effects of SAC have not been determined in ovarian cancer cells. Here, we developed a novel method for producing native SAC in Escherichia coli using a small ubiquitin-related modifier (SUMO) fusion system. This fusion system not only greatly improved the solubility of target protein but also enhanced the expression level of SUMO-SAC. After purified by Ni-NTA affinity chromatography, SUMO tag was cleaved from SUMO-SAC fusion protein using SUMO protease to obtain recombinant SAC. Furthermore, we simplified the purification process by combining the SUMO-SAC purification and SUMO tag cleavage into one step. Finally, the purity of recombinant SAC reached as high as 95% and the yield was 25 mg/L. Our results demonstrated that recombinant SAC strongly inhibited proliferation and induced apoptosis in ovarian cancer cells SKOV-3. Immunofluorescence analysis and competitive binding reaction showed that recombinant SAC could specifically induce apoptosis of SKOV-3 cells through combination with cell surface receptor, GRP78. Therefore, we have developed an effective strategy for expressing bioactive SAC in prokaryotic cells, which supports the application of SAC in ovarian cancer therapy.

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
Fig. 6

Similar content being viewed by others

References

  1. Hebbar, N., Wang, C., & Rangnekar, V. M. (2012). Mechanisms of apoptosis by the tumor suppressor Par-4. Journal of Cellular Physiology, 227, 3715–3721.

    Article  CAS  Google Scholar 

  2. El-Guendy, N., & Rangnekar, V. M. (2003). Apoptosis by Par-4 in cancer and neurodegenerative diseases. Experimental Cell Research, 283, 51–66.

    Article  CAS  Google Scholar 

  3. El-Guendy, N., Zhao, Y., Gurumurthy, S., Burikhanov, R., & Rangnekar, V. M. (2003). Identification of a unique core domain of par-4 sufficient for selective apoptosis induction in cancer cells. Molecular and Cellular Biology, 23, 5516–5525.

    Article  CAS  Google Scholar 

  4. Zhao, Y., & Rangnekar, V. M. (2008). Apoptosis and tumor resistance conferred by Par-4. Cancer Biology & Therapy, 7, 1867–1874.

    Article  CAS  Google Scholar 

  5. Burikhanov, R., Zhao, Y., Goswami, A., Qiu, S., Schwarze, S. R., & Rangnekar, V. M. (2009). The tumor suppressor Par-4 activates an extrinsic pathway for apoptosis. Cell, 138, 377–388.

    Article  CAS  Google Scholar 

  6. Zhao, Y., Burikhanov, R., Brandon, J., Qiu, S., Shelton, B. J., Spear, B., Bondada, S., Bryson, S., & Rangnekar, V. M. (2011). Systemic Par-4 inhibits non-autochthonous tumor growth. Cancer Biology & Therapy, 12, 152–157.

    Article  CAS  Google Scholar 

  7. Shrestha-Bhattarai, T., & Rangnekar, V. M. (2010). Cancer-selective apoptotic effects of extracellular and intracellular Par-4. Oncogene, 29, 3873–3880.

    Article  CAS  Google Scholar 

  8. Sayers, T. J. (2011). Targeting the extrinsic apoptosis signaling pathway for cancer therapy. Cancer Immunology, Immunotherapy, 60, 1173–1180.

    Article  CAS  Google Scholar 

  9. Chaudhry, P., Singh, M., Parent, S., & Asselin, E. (2012). Prostate apoptosis response 4 (Par-4), a novel substrate of caspase-3 during apoptosis activation. Molecular and Cellular Biology, 32, 826–839.

    Article  CAS  Google Scholar 

  10. Braun, P., Hu, Y., Shen, B., Halleck, A., Koundinya, M., Harlow, E., & LaBaer, J. (2002). Proteome-scale purification of human proteins from bacteria. Proceedings of the National Academy of Sciences of the United States of America, 99, 2654–2659.

    Article  CAS  Google Scholar 

  11. Zafar, A., Aftab, M. N., ud Din, Z., Aftab, S., Iqbal, I., & ul Haq, I. (2016). Cloning, purification and characterization of a highly thermostable amylase gene of Thermotoga petrophila into Escherichia coli. Applied Biochemistry and Biotechnology, 178, 831–848.

    Article  CAS  Google Scholar 

  12. Baneyx, F. (1999). Recombinant protein expression in Escherichia coli. Current Opinion in Biotechnology, 10, 411–421.

    Article  CAS  Google Scholar 

  13. Mahamad, P., Boonchird, C., & Panbangred, W. (2016). High level accumulation of soluble diphtheria toxin mutant (CRM197) with co-expression of chaperones in recombinant Escherichia coli. Applied Microbiology and Biotechnology, 100, 6319–6330.

    Article  CAS  Google Scholar 

  14. Tian, H., Zhao, Y., Chen, N., Wu, M., Gong, W., Zheng, J., Fernig, D. G., Jungbauer, A., Wang, D., Li, X., & Jiang, C. (2016). High production in E. coli of biologically active recombinant human fibroblast growth factor 20 and its neuroprotective effects. Applied Microbiology and Biotechnology, 100, 3023–3034.

    Article  CAS  Google Scholar 

  15. Rueda, F., Cano-Garrido, O., Mamat, U., Wilke, K., Seras-Franzoso, J., García-Fruitós, E., & Villaverde, A. (2014). Production of functional inclusion bodies in endotoxin-free Escherichia coli. Applied Microbiology and Biotechnology, 98, 9229–9238.

    Article  CAS  Google Scholar 

  16. Gao, J., & Wang, H. (2015). Prokaryotic expression, refolding and purification of high-purity mouse Midkine in Escherichia coli. Applied Biochemistry and Biotechnology, 176, 454–466.

    Article  CAS  Google Scholar 

  17. Kong, B., & Guo, G. L. (2011). Enhanced in vitro refolding of fibroblast growth factor 15 with the assistance of SUMO fusion partner. PloS One, 6, e20307.

    Article  CAS  Google Scholar 

  18. Fan, J., Huang, L., Sun, J., Qiu, Y., Zhou, J., & Shen, Y. (2015). Strategy for linker selection to enhance refolding and bioactivity of VAS-TRAIL fusion protein based on inclusion body conformation and activity. Journal of Biotechnology, 209, 16–22.

    Article  CAS  Google Scholar 

  19. Kapust, R. B., & Waugh, D. S. (1999). Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Science, 8, 1668–1674.

    Article  CAS  Google Scholar 

  20. Li, Y. (2013). Production of human antimicrobial peptide LL-37 in Escherichia coli using a thioredoxin-SUMO dual fusion system. Protein Expression and Purification, 87, 72–78.

    Article  CAS  Google Scholar 

  21. Zhang, M., Qiu, Z., Li, Y., Yang, Y., Zhang, Q., Xiang, Q., Su, Z., & Huang, Y. (2013). Construction and characterization of a recombinant human beta defensin 2 fusion protein targeting the epidermal growth factor receptor: in vitro study. Applied Microbiology and Biotechnology, 97, 3913–3923.

    Article  CAS  Google Scholar 

  22. Dian, C., Eshaghi, S., Urbig, T., McSweeney, S., Heijbel, A., Salbert, G., & Birse, D. (2002). Strategies for the purification and on-column cleavage of glutathione-S-transferase fusion target proteins. Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, 769, 133–144.

    Article  CAS  Google Scholar 

  23. Chaubey, N., & Ghosh, S. S. (2013). Molecular cloning, purification and functional implications of recombinant GST tagged hGMCSF cytokine. Applied Biochemistry and Biotechnology, 169, 1713–1726.

    Article  CAS  Google Scholar 

  24. Ichikawa, Y., Kagawa, W., Saito, K., Chikashige, Y., Haraguchi, T., Hiraoka, Y., & Kurumizaka, H. (2013). Purification and characterization of the fission yeast telomere clustering factors, Bqt1 and Bqt2. Protein Expression and Purification, 88, 207–213.

    Article  CAS  Google Scholar 

  25. Ye, T., Lin, Z., & Lei, H. (2008). High-level expression and characterization of an anti-VEGF165 single-chain variable fragment (scFv) by small ubiquitin-related modifier fusion in Escherichia coli. Applied Microbiology and Biotechnology, 81, 311–317.

    Article  CAS  Google Scholar 

  26. Li, Y. (2013). Recombinant production of crab antimicrobial protein scygonadin expressed as thioredoxin and SUMO fusions in Escherichia coli. Applied Biochemistry and Biotechnology, 169, 1847–1857.

    Article  CAS  Google Scholar 

  27. Upadhyay, S. K., Saurabh, S., Rai, P., Singh, R., Chandrashekar, K., Verma, P. C., Singh, P. K., & Tuli, R. (2010). SUMO fusion facilitates expression and purification of garlic leaf lectin but modifies some of its properties. Journal of Biotechnology, 146, 1–8.

    Article  CAS  Google Scholar 

  28. Xu, R., Dong, Y., Wang, L., Tao, X., Sun, A., & Wei, D. (2014). TAT-RhoGDI2, a novel tumor metastasis suppressor fusion protein: expression, purification and functional evaluation. Applied Microbiology and Biotechnology, 98, 9633–9641.

    Article  CAS  Google Scholar 

  29. Lv, X., Zhang, J., Xu, R., Dong, Y., Sun, A., Shen, Y., & Wei, D. (2016). Gigantoxin-4-4D5 scFv is a novel recombinant immunotoxin with specific toxicity against HER2/neu-positive ovarian carcinoma cells. Applied Microbiology and Biotechnology, 100, 6403–6413.

    Article  CAS  Google Scholar 

  30. Lee, T. J., Jang, J. H., Noh, H. J., Park, E. J., Choi, K. S., & Kwon, T. K. (2010). Overexpression of Par-4 sensitizes TRAIL-induced apoptosis via inactivation of NF-κB and Akt signaling pathways in renal cancer cells. Journal of Cellular Biochemistry, 109, 885–895.

    CAS  Google Scholar 

  31. Lee, T. J., Lee, J. T., Kim, S. H., Choi, Y. H., Song, K. S., Park, J. W., & Kwon, T. K. (2008). Overexpression of Par-4 enhances thapsigargin-induced apoptosis via downregulation of XIAP and inactivation of Akt in human renal cancer cells. Journal of Cellular Biochemistry, 103, 358–368.

    Article  CAS  Google Scholar 

  32. Jagtap, J. C., Dawood, P., Shah, R. D., Chandrika, G., Natesh, K., Shiras, A., Hegde, A. S., Ranade, D., & Shastry, P. (2014). Expression and regulation of prostate apoptosis response-4 (Par-4) in human glioma stem cells in drug-induced apoptosis. PloS One, 9, e88505.

    Article  Google Scholar 

  33. Meynier, S., Kramer, M., Ribaux, P., Tille, J. C., Delie, F., Petignat, P., & Cohen, M. (2015). Role of PAR-4 in ovarian cancer. Oncotarget, 6, 22641–22652.

    Article  Google Scholar 

  34. Kline, C. L., & Irby, R. B. (2011). The pro-apoptotic protein prostate apoptosis response protein-4 (Par-4) can be activated in colon cancer cells by treatment with Src inhibitor and 5-FU. Apoptosis, 16, 1285–1294.

    Article  CAS  Google Scholar 

  35. Zhang, X. X., Li, H. D., Zhao, L., Song, H. J., Wang, G., Guo, Q. J., Luan, Z. D., & Su, R. J. (2013). The cell surface GRP78 facilitates the invasion of hepatocellular carcinoma cells. BioMed Research International, 2013, 917296.

    Google Scholar 

  36. Li, Z., Zhang, L., Zhao, Y., Li, H., Xiao, H., Fu, R., Zhao, C., Wu, H., & Li, Z. (2013). Cell-surface GRP78 facilitates colorectal cancer cell migration and invasion. The International Journal of Biochemistry & Cell Biology, 45, 987–994.

    Article  CAS  Google Scholar 

  37. Irby, R. B., & Kline, C. L. (2013). Par-4 as a potential target for cancer therapy. Expert Opinion on Therapeutic Targets, 17, 77–87.

    Article  CAS  Google Scholar 

Download references

Funding

This work was funded by the National Natural science Foundation of China (No. 21646005/B060806) and China Postdoctoral Science Foundation funded project (No. 2016M601529).

Author information

Authors and Affiliations

  1. State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, China

    Jian Zhang, Aiyou Sun, Yuguo Dong & Dongzhi Wei

Authors
  1. Jian Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Aiyou Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yuguo Dong
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dongzhi Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Aiyou Sun or Dongzhi Wei.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, J., Sun, A., Dong, Y. et al. Recombinant Production and Characterization of SAC, the Core Domain of Par-4, by SUMO Fusion System. Appl Biochem Biotechnol 184, 1155–1167 (2018). https://doi.org/10.1007/s12010-017-2599-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-017-2599-9

Keywords

Search

Navigation